Biosensors, extracorporeal devices and methods for detecting substances using crosslinked protein crystals

ABSTRACT

Proteins such as enzymes and antibodies are immobilized by crosslinking crystals of the proteins such as microcrystals having a cross-section of 10 -1  mm or less with a multifunctional crosslinking agent. The crosslinked protein crystals may be lyophilized for storage. Crystals of an enzyme such as thermolysin, elastase, asparaginase, lysozyme, lipase or urease may be crosslinked to provide crosslinked enzyme crystals that retain at least 91% activity after incubation for three hours in the presence of a concentration of Pronase™ that causes the soluble uncrosslinked form of the enzyme to lose at least 94% of its initial activity under the same conditions. A preferred Pronase™:enzyme ratio is 1:40. Crosslinked enzyme or antibody crystals may be used in an assay, diagnostic kit or biosensor for detecting an analyte, in an extracorporeal device for altering a component of a fluid, in producing a product such as using crosslinked thermolysin crystals to produce aspartame, in separating a substance from a mixture, and in therapy.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 08/017,510,filed Feb. 12, 1993, now U.S. Pat. No. 5,618,710, which is acontinuation-in-part of application Ser. No. 07/864,424, filed Apr. 6,1992, now abandoned, which is a continuation-in-part of application Ser.No. 07/720,237, filed Jun. 24, 1991, now abandoned, which is acontinuation-in-part of application Ser. No. 07/562,280, filed Aug. 3,1990, now abandoned. The teachings of Ser. Nos. 07/864,424, 07/720,237and 07/562,280 are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Enzymes are used as industrial catalysts for the large and laboratoryscale economical production of fine and specialty chemicals (Jones, J.B., Tetrahedron 42: 3351-3403 (1986)), for the production of foodstuffs(Zaks et. al., Trends in Biotechnology 6: 272-275 (1988)), and as toolsfor the synthesis of organic compounds (Wong, C.-H., Science 244:1145-1152 (1989); CHEMTRACTS-Org. Chem. 3: 91-111 (1990); Klibanov, A.M., Acc. Chem. Res. 23: 114-120 (1990)).

Enzyme-based manufacturing can significantly reduce the environmentalpollution burden implicit in the large scale manufacturing of otherwiseunusable chemical intermediates, as shown in the large scale productionof acrylamide using the enzyme, nitrile hydratase (Nagasawa, T. andYamada, H., Trends in Biotechnology 7: 153-158 (1989)).

Enzymes are also used in biosensor applications to detect varioussubstances of clinical, industrial and other interest (Hall, E.,"Biosensors", Open University Press (1990)). In the clinical area,enzymes may be used in extracorporeal therapy, such as hemodialysis andhemofiltration, where the enzymes selectively remove waste and toxicmaterials from blood (Klein, M. and Langer, R., Trends in Biotechnology4: 179-185 (1986)). Enzymes are used in these areas because theyfunction efficiently as catalysts for a broad range of reaction types,at modest temperatures, and with substrate specificity andstereoselectivity. Nonetheless, there are disadvantages associated withthe use of soluble enzyme catalysts which have limited their use inindustrial and laboratory chemical processes (Akiyama et. al., CHEMTECH627-634 (1988)).

Enzymes are expensive and relatively unstable compared to mostindustrial and laboratory catalysts, even when they are used in aqueousmedia where enzymes normally function. Many of the more economicallyinteresting chemical reactions carried out in common practice areincompatible with aqueous media, where, for example, substrates andproducts are often insoluble or unstable, and where hydrolysis cancompete significantly. In addition, the recovery of soluble enzymecatalyst from product and unreacted substrate in the feedstock oftenrequires the application of complicated and expensive separationtechnology. Finally, enzymes are difficult to store in a manner thatretains their activity and functional integrity, for commerciallyreasonable periods of time (months to years) without having to resort torefrigeration (4° C. to -80° C. to liquid N₂ temperatures), or tomaintenance in aqueous solvents of suitable ionic strength, pH, etc.

Enzyme immobilization methods have, in many instances, circumventedthese disadvantages. Immobilization can improve the stability of enzymecatalysts and protect their functional integrity in the harsh solventenvironments and extreme temperatures characteristic of industrial andlaboratory chemical processes (Hartmeier, W., Trends in Biotechnology 3:149-153 (1985)). Continuous flow processes may be operated withimmobilized enzyme particles in columns, for example, where the solublefeedstock passes over the particles and is gradually converted intoproduct. As used herein, the term enzyme immobilization refers to theinsolubilization of enzyme catalyst by attachment to, encapsulation of,or by aggregation into macroscopic (10⁻¹ mm) particles.

A number of useful reviews of enzyme immobilization methods haveappeared in the literature (Maugh, T. H., Science 223: 474-476 (1984);Tramper, J., Trends in Biotechnology 3: 45-50 (1985)). Maugh describesfive general approaches to the immobilization of enzymes. These include:adsorption on solid supports (such as ion-exchange resins); covalentattachments to supports (such as ion-exchange resins, porous ceramics orglass beads); entrapment in polymeric gels; encapsulation; and theprecipitation of soluble proteins by cross-linking them withbifunctional reagents in a random and undefined manner. In addition, onecan immobilize whole cells (usually dead and made permeable) which haveexpressed the desired enzyme activity at high levels (e.g., Nagasawa, T.and Yamada, H., Trends in Biotechnology 7: 153-158 (1989)).

Each of these immobilization procedures has its own advantages andlimitations and none can be considered optimal or dominating. In most ofthem, the enzyme catalyst ultimately represents only a small fraction ofthe total volume of material present in the chemical reactor. As such,the bulk of the immobilized medium is made up of inert, but often costlycarrier material. In all of them, the immobilizing interactions of theenzyme catalyst molecules with each other and/or with the carriermaterial tend to be random and undefined. As a result, although theseinteractions confer some enhanced stability to the enzyme catalystmolecules, their relative non-specificity and irregularity makes thatstabilization sub-optimal and irregular. In most cases, access to theactive site of the enzyme catalyst remains ill-defined. In addition, theimmobilization methods described above fail to deal with problemsassociated with storage and refrigeration. Nor can conventionallyimmobilized enzymes generally be manipulated, as in being exchanged intoone or another solvent of choice, without risk to the structural andfunctional integrity of the enzyme. In practical terms, except for theattached tether to the carrier particle, conventionally immobilizedenzymes bear close resemblance to soluble enzymes, and share with them asusceptibility to denaturation and loss of function in harshenvironments. In general, immobilization methods lead to a reduction ofobserved enzyme-catalyzed reaction rates relative to those obtained insolution. This is mostly a consequence of the limits of inward diffusionof substrate and outward diffusion of product within the immobilizedenzyme particle (Quiocho, F. A., and Richards, F. M., Biochemistry 5:4062-4076 (1967)). The necessary presence of inert carrier in theimmobilized enzyme particles increases the mean free path between thesolvent exterior of the immobilized enzyme particle and the active siteof the enzyme catalyst and thus exacerbates these diffusion problems.When dealing with immobilized cells, the diffusion problem isparticularly severe, even if cell walls and membranes are made permeableto substrate and product in some way. One would further be concernedwith the multitude of contaminating enzymatic activities, metabolites,and toxins contained in cells, and with the stability of cells in harshsolvents or extreme temperature operating environments. An improvedimmobilization technique which avoids the limitations of the presentlyavailable methods would be helpful in promoting the use of enzymes asindustrial catalysts, particularly if it were shown to be useful on alarge scale (Daniels, M. J., Methods in Enzymology 136: 371-379 (1987)).

SUMMARY OF THE INVENTION

The present invention relates to a method of immobilizing a protein,particularly an enzyme or an antibody, by forming crystals of the enzymeor antibody and, generally, also crosslinking the resulting crystalsthrough use of a bifunctional reagent; crosslinked immobilized enzymecrystals (referred to as CLECs or CLIECs) made by this method;crosslinked immobilized antibody crystals (referred to as CLACs); thelyophilization of the resulting crystals as a means of improving thestorage, handling, and manipulation properties of immobilized enzymesand antibodies; a method of making a desired product by means of areaction catalyzed by a CLEC or a set of CLECs; and methods in which theCLACs of the present invention are used, such as a method of separatingor purifying a substance or molecule of interest, in which a CLAC whichrecognizes (binds) the substance or molecule of interest serves as animmunospecific reagent. In another embodiment, a CLAC can be used fordetection of a substance or molecule of interest in a sample, such as abiological sample, water, or other sample; this embodiment is useful,for example, for diagnostic purposes. In a further embodiment, CLACs ofthe present invention can be used for therapeutic purposes, in much thesame manner monoclonal antibodies are now used therapeutically; in manyinstances, a CLAC of a particular enzyme can simply replace orsubstitute for presently-used (non-CLAC) antibodies. A particularadvantage to CLACs is their enhanced resistance to degradation (e.g.,enhanced protease resistance), relative to that of non-CLAC antibodies.

In the method of the present invention by which enzyme crystals areproduced, small protein crystals (crystals of approximately 10⁻¹ mm insize) are grown from aqueous solutions, or aqueous solutions containingorganic solvents, in which the enzyme catalyst is structurally andfunctionally stable. In a preferred embodiment, crystals are thencrosslinked with a bifunctional reagent, such as glutaraldehyde. Thiscrosslinking results in the stabilization of the crystal latticecontacts between the individual enzyme catalyst molecules constitutingthe crystal. As a result of this added stabilization, the crosslinkedimmobilized enzyme crystals can function at elevated temperatures,extremes of pH and in harsh aqueous, organic, or near-anhydrous media,including mixtures of these. That is, a CLEC of the present inventioncan function in environments incompatible with the functional integrityof the corresponding uncrystallized, uncrosslinked, native enzyme orconventionally immobilized enzyme catalysts. CLACs can be made in asimilar manner, using commercially available antibodies or antibodiesproduced against a specific antigen or hapten; entire antibodies orantibody fragments (e.g., FAb fragments) can be used to produce acorresponding CLAC.

In addition, CLECs made by this method can be subjected tolyophilization, producing a lyophilized CLEC which can be stored in thislyophilized form at non-refrigerated (room) temperatures for extendedperiods of time, and which can be easily reconstituted in aqueous,organic, or mixed aqueous-organic solvents of choice, without theformation of amorphous suspensions and with minimal risk ofdenaturation.

The present invention also relates to CLECs produced by the presentmethod and to their use in laboratory and large scale industrialproduction of selected materials, such as chiral organic molecules,peptides, carbohydrates, lipids, or other chemical species. Presently,these are typically prepared by conventional chemical methods, which mayrequire harsh conditions (e.g. aqueous, organic or near-anhydroussolvents, mixed aqueous/organic solvents or elevated temperatures) thatare incompatible with the functional integrity of uncrystallized,uncrosslinked, native enzyme catalyst. Other macromolecules withcatalytic activity can also be incorporated into the proposed CLECtechnology. These might include catalytic antibodies (Lerner, R. A.,Benkovic, S. J., and Schultz, P. G., Science 252:659-667 (1991)) andcatalytic polynucleotides (Cech, T. R., Cell 64:667-669 (1991);Celander, D. W., and Cech, T. R. Science, 251:401-407 (1991)).

The present invention also relates to a method of making a selectedproduct by means of a reaction catalyzed by a CLEC of the presentinvention.

In an example of the method and practice of the present invention, theenzyme thermolysin, a zinc metalloprotease, was used to synthesize achiral precursor of the dipeptidyl artificial sweetener, aspartame.(Example 1) The enzyme thermolysin was crystallized from a startingaqueous solution of 45% dimethyl sulfoxide, and 55% 1.4M calciumacetate, 0.05M sodium cacodylate, pH 6.5. The resulting crystals werecross-linked with glutaraldehyde to form a thermolysin CLEC. Thethermolysin CLEC was then transferred from the aqueous crystallizationsolution in which it was made, into a solution of ethyl acetatecontaining the substrates, N-(benzyloxycarbonyl)-L-aspartic acid(Z-L-Asp) and L-phenylalanine methyl ester (L-Phe-OMe). The thermolysinCLEC was then used to catalyze a condensation reaction of the twosubstrates to synthesizeN-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester(Z-L-Asp-L-Phe-OMe), which is the dipeptidyl precursor of the artificialsweetener aspartame. Using any one of many known techniques (see, e.g.Lindeberg, G., J. Chem Ed. 64: 1062-1064 (1987)) the L-aspartic acid inthe synthesized dipeptidyl precursor can be deprotected by the removalof the benzyloxycarbonyl (Z-) group to produce aspartame(L-Asp-L-Phe-OMe).

In a second example of the method and practice of the present invention,the enzyme thermolysin was used to produce thermolysin CLECs. Theactivity and stability of thermolysin CLECs were compared to that ofsoluble thermolysin under optimum conditions and conditions of extremepH and temperature, following incubation in the presence of organicsolvents and following incubation in the presence of exogenous protease.(Example 2) See also Example 3, which describes further work withthermolysin CLEC.

The enzyme thermolysin was crystallized from a solution of 1.2M calciumacetate and 30% dimethyl sulfoxide pH 8.0. The resulting crystals werecrosslinked with glutaraldehyde at a concentration of 12.5% to form athermolysin CLEC. The thermolysin CLEC was then lyophilized by astandard procedure (Cooper, T. G., The Tools of Biochemistry, pp.379-380 (John Wiley and Sons, New York (1977)) to form a lyophilizedenzyme CLEC of thermolysin. This lyophilized CLEC was then transformeddirectly into the different aqueous, organic, and mixed aqueous/organicsolvents of choice without an intervening solvent exchange procedure,without formation of amorphous suspensions, and with minimal risk ofdenaturation. These solvents included acetonitrile, dioxane, acetone,and tetrahydrofuran, but not to the exclusion of others. Followingincubation, activity was assayed spectrophotometrically by cleavage ofthe dipeptide substrate FAGLA (furylacryloyl-glycyl-L-leucine amide).

In a third example of the method and practice of the present invention,the enzyme elastase (porcine pancreatic) was crystallized from anaqueous solution of 5.5 mg/ml protein in 0.1 M sodium acetate at pH 5.0at room temperature (Sawyer, L. et al., J. Mol. Biol. 118:137-208). Theresulting crystals were crosslinked with glutaraldehyde at aconcentration of 5% to form an elastase CLEC. (Example 4) The elastaseCLEC was lyophilized as described in Example 2.

In a fourth example of the method and practice of the present invention,and as disclosed here, the enzyme esterase (porcine liver) wascrystallized from an aqueous solution of 15 mg/ml protein in 0.25 Mcalcium acetate at pH 5.6 at room temperature. The resulting crystalswere crosslinked with glutaraldehyde at a concentration of 12.5% to forman esterase CLEC. (Example 5) The esterase CLEC was lyophilized asdescribed in Example 2.

In a fifth example of the method and practice of the present invention,and as disclosed here, the enzyme lipase (Geotrichum candidum) wascrystallized from an aqueous solution of 20 mg/ml protein in 50 mM Trisat pH 7 at room temperature. The resulting crystals were crosslinkedwith glutaraldehyde at a concentration of 12.5% to form a lipase CLEC.The lipase CLEC was lyophilized as described in Example 2. In addition,Candida cylindracea lipase has been crystallized and crosslinked, asdescribed herein; the resulting CLEC was shown to retain significantenzymatic activity. Further, porcine pancreatic lipase has beencrystallized and cross-linked; preliminary assessment of the resultingCLEC showed that it retained less activity than either of the otherlipases (G. candidum or C. cylindracea). (See Examples 6, 10 and 11).

In a sixth example of the method and practice of the present invention,the enzyme lysozyme (hen egg white) was crystallized from an aqueoussolution of 40 mg/ml protein in 40 mM sodium acetate buffer containing5% sodium chloride at pH 7.4 at room temperature (Blake, C. C. F. etal., Nature, 196:1173 (1962)). The resulting crystals were crosslinkedwith glutaraldehyde at a concentration of 20% to form a lysozyme CLEC.(Example 7) The lysozyme CLEC was lyophilized as described in Example 2.

In a seventh example of the method and practice of the presentinvention, the enzyme asparaginase (Escherichia coli) was crystallizedfrom an aqueous solution of 25 mg/ml protein in 50 mM sodium acetate and33% ethanol at pH 5.0 at 4° C. The crystallization is a modification ofthe procedure described by Grabner et al. [U.S. Pat. No. 3,664,926(1972)]. As disclosed here, the resulting crystals were crosslinked withglutaraldehyde at a concentration of 7.5% to form an asparaginase CLEC.(Example 8) The asparaginase CLEC was lyophilized as described inExample 2.

In an eighth example of the method and practice of the presentinvention, the enzyme urease (Jack Bean) was crystallized and theresulting urease crystals were crosslinked. (Example 9) The urease CLECwas lyophilized, as described in Example 2. Enzymatic activity of theurease CLEC was compared with that of soluble urease. In addition,enzymatic activity of urease CLEC in aqueous buffer was compared withactivity in sera, a biologically relevant medium; activity in sera wascomparable to activity in aqueous buffer.

Other enzymes which can be immobilized in a similar manner and used tocatalyze an appropriate reaction include luciferase. Other enzymes, suchas those listed in Tables 1-5, can also be crystallized and crosslinkedusing the present method, to produce a desired CLEC which can, in turn,be used to catalyze a reaction which results in production of a selectedproduct or to catalyze a reaction which is an intermediate step (i.e.one in a series of reactions) in the production of a selected product.It is recognized that although crosslinking helps stabilize the majorityof crystals, it is neither necessary nor desirable in all cases. Somecrystalline enzymes retain functional and structural integrity in harshenvironments even in the absence of crosslinking. Although in thepreferred embodiment, the crystal is crosslinked, crosslinking is notalways necessary to produce an enzyme crystal useful in the presentmethod.

CLECs have several key characteristics that confer significantadvantages over conventional enzyme immobilization methods presently inuse. CLECs dispense with the need for a separate, inert supportstructure. Lack of an inert support will improve substrate and productdiffusion properties within CLECs and provides enzyme concentrationswithin the crystal that are close to the theoretical packing limit formolecules of such size. High enzyme concentrations can lead tosignificant operational economies through the increased effectiveactivity of a given volume of catalyst, reduction in substrate contacttime with enzyme and overall reduction in plant size and capital costs(Daniels, M. J., Methods in Enzymol. 136: 371-379 (1987)). Theuniformity across crystal volume and enhanced stability of theconstituent enzyme in CLECs creates novel opportunities for the use ofenzyme catalysis in harsh conditions, such as elevated temperature, andaqueous, organic or near-anhydrous solvents, as well as mixtures ofthese. In addition, the restricted solvent access and regular proteinenvironment implicit in a crystal lattice should lead to improved metalion and co-factor retention for CLECs vs. conventional immobilizedenzyme systems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of results of assessment of enzymaticactivity of soluble and thermolysin CLEC.

FIG. 2 is a graphic representation of results of a comparison of pHdependencies of thermolysin CLEC and soluble thermolysin.

FIG. 3 is a graphic representation of measurement of the activity ofsoluble and crystalline thermolysin after incubation at 65° C.

FIG. 4 is a graphic representation of results of assessment ofresistance of soluble and thermolysin CLEC to exogenous proteolyticdegradation.

FIG. 5 is a series of graphic representations (A-C) of results ofassessment of continuous batch synthesis of the aspartame precursorusing soluble thermolysin and CLEC thermolysin.

FIG. 6 is a graphic representation of results of the assessment ofenzymatic activity for soluble elastase and the corresponding elastaseCLEC.

FIG. 7 is a graphic representation of the resistance of soluble elastaseand the corresponding elastase CLEC to exogenous proteolyticdegradation.

FIG. 8 is a graphic representation of results of the assessment ofenzymatic activity for soluble esterase and the corresponding esteraseCLEC.

FIG. 9 is a graphic representation of the resistance of soluble esteraseand the corresponding esterase CLEC to exogenous proteolyticdegradation.

FIG. 10 is a graphic representation of results of the assessment ofenzymatic activity for soluble lipase and the corresponding lipase CLEC.

FIG. 11 is a graphic representation of results of the assessment ofenzymatic activity for soluble lysozyme and the corresponding lysozymeCLEC.

FIG. 12 is a graphic representation of results of the assessment ofenzymatic activity for soluble asparaginase and the correspondingasparaginase CLEC.

FIG. 13 is a graphic representation of results of the assessment ofenzymatic activity of soluble urease and the corresponding urease CLEC.

FIG. 14 is a graphic representation of results of assessment of pHdependence and stability of soluble urease and the corresponding ureaseCLEC.

FIG. 15 is a graphic representation of results of assessment of thermalstability of soluble urease and the corresponding urease CLEC.

FIG. 16 is a graphic representation of results of assessment ofresistance to exogenous proteolysis of soluble urease and thecorresponding urease CLEC.

FIG. 17 is a graphic representation of results of assessment of ureaseCLEC enzymatic activity in aqueous buffer and in sera.

DETAILED DESCRIPTION OF THE INVENTION

A simple, general procedure that assures stability and function for agiven enzyme or set of enzymes under conditions which are of interest tothe synthetic chemist and which are too harsh for use with enzymes usingpresently available methods, would be very useful. Cross-linkedimmobilized enzyme crystals (referred to as CLECs or CLIECs) asdescribed here can be used for this purpose. Stabilization of thecrystal lattice and of the constituent enzyme catalysts in the crystalby the cross-linking reaction permits the use of CLECs in environments,including aqueous, organic or near-anhydrous solvents, mixtures of thesesolvents, extremes of pH and elevated temperatures, which areincompatible with enzyme function using presently available methods. Inaddition, the stabilization of the crystal lattice in CLECs makespossible the lyophilization of CLECs by standard methods. LyophilizedCLECs can be stored for commercially attractive periods of time (monthsto years) in the absence of refrigeration, and facilitate the rapid anduncomplicated utilization of CLECs in industrial and laboratory scaleprocesses by the simple addition of solvents of choice, without need forintervening solvent exchange processes. CLECs are also highly resistantto digestion by exogenous proteases. The method of the present inventionfacilitates the use of versatile enzyme catalysts in mainstreamindustrial chemical processes, as well as in the laboratory synthesis ofnovel compounds for research.

Although crosslinking contributes to the stability of a crystal enzyme,it is neither necessary nor desirable in all cases. Some crystallizedenzymes retain functional and structural integrity in harsh environmentseven in the absence of crosslinking. The preferred embodiment of thepresent method includes cross-linking of a crystal enzyme and isdescribed in detail in the following sections. It is to be understood,however, that crystallized enzymes which are not subsequentlycross-linked can be used in some embodiments of the present invention.

The regular interactions between the constituent enzyme molecules in thecrystal lattice of a CLEC result in well defined pores of limited sizeleading to the enzyme molecules within the body of a CLEC. As a result,substrates larger than the available pore size will not penetrate thebody of the CLEC particle.

As a consequence of the limited pore size, many enzymatic reactions ofcommercial and academic interest involving substrates larger than thepore size of the CLECs would be beyond the scope of the presentinvention. This would include most reactions involving large polymers,such as proteins, polynucleotides, polysaccharides, and other organicpolymers, where the number of polymeric subunits would be such as tomake the polymer larger than the crystal pore size in CLECs. In suchinstances, however, catalysis can still take place on the CLEC surface.

The present invention is a method of immobilizing a selected protein,particularly an enzyme, by crystallizing and crosslinking the protein,resulting in production of a crosslinked immobilized enzyme crystal(CLEC) which can be used to catalyze production of a selected product,such as a peptide, carbohydrate, lipid or chiral organic molecule. Theselected product can be produced by altering a single substrate (e.g.,to produce a breakdown product or other product) or by combining thesubstrate with an additional substance or substances (e.g., a secondsubstrate, molecule or compound to be added through the action of theCLEC). The present invention further relates to such CLECs and to amethod of making a selected product by means of a CLEC-catalyzedreaction or CLEC-catalyzed step in a series of reactions. In oneembodiment of the present invention, the dipeptidyl precursor ofaspartame has been produced in a condensation reaction catalyzed bycross-linked immobilized thermolysin made by the present method. Inanother embodiment of this invention, the indicator substrate, FAGLA,has been cleaved to produce a colorimetric product, whose presence isindicative of enzyme activity in a thermolysin CLEC. FAGLA hydrolysishas been used as a model reaction to indicate the robustness of thethermolysin CLEC in a number of environments that would be normallyincompatible with that enzyme's activity.

In other embodiments of this invention, the enzymes elastase, esterase,lipase, asparaginase, and lysozyme have been used to cleave variousindicated substances, such as p-nitrophenyl acetate (esterase andlipase), succinyl-(ala)3-p-nitroanilide (elastase), 4-methylumbelliferylN-acetyl-chitrioside (lysozyme) and NADH (asparaginase) and urea(urease).

By the method of this invention, one of ordinary skill in the art canadapt a protocol for making a desired product by means of a reactioncatalyzed by an immobilized enzyme. The enzyme of interest, whencrystallized from an appropriate solution, can be cross-linked withglutaraldehyde or other suitable bifunctional reagent in thecrystallization solution to produce a CLEC of that enzyme. Subsequently,the CLEC of the enzyme of choice can be lyophilized as described inExample 2.

There are several advantages which the use of a CLEC offers overpresently-available enzyme-catalyzed methods. For example, thecross-linked crystal matrix in a CLEC provides its own support.Expensive carrier beads, glasses, gels, or films are not required inorder to tie down the enzyme catalyst, as they are inpresently-available immobilization methods. As a result, theconcentration of enzyme in a CLEC is close to the theoretical packinglimit that can be achieved for molecules of a given size, greatlyexceeding densities achievable even in concentrated solutions. Theentire CLEC consists of active enzyme (and not inactive carrier), andthus, the diffusion-related reduction of enzyme reaction rates usuallyobserved with conventionally immobilized enzymes relative to enzymes insolution should be minimized, since the mean free path for substrate andproduct between active enzyme and free solvent will be greatly shortenedfor CLECs (compared to a conventional immobilized enzyme carrierparticles). These high protein densities will be particularly useful inbiosensor, analytical and other applications requiring large amounts ofprotein in small volumes. In industrial processes, the superiorperformance and compactness of CLECs results in significant operatingeconomies, by increasing the effective activity of a given volume ofcatalyst, thereby allowing reductions in plant size, as well as capitalcosts (Daniels, M. J., Methods in Enzymol. 136: 371-379 (1987)). CLECsare relatively monodisperse, with a macroscopic size and shapereflecting natural crystal growth characteristics of the individualenzyme catalysts. Replacement of existing carrier-immobilized enzymemedia with CLECs should not be difficult, since both systems arecomparable in size and shape, and both can be similarly recovered fromfeedstock by any number of simple methods, including basic economicaloperations such as filtration, centrifugation, decantation of solvent,and others.

In addition, the use of lyophilized CLECs permits routine handling andstorage of these materials prior to use (dry storage at room temperaturewithout refrigeration, for extended periods of time). Lyophilized CLECsalso allow for routine formulation by direct addition of solvents andsubstrates of interest, without lengthy solvent exchange processes, orthe formation of amorphous suspensions. The lyophilized CLEC formextends the general utility of the enzymes as catalysts to a broaderspectrum of enzymes and functional conditions.

A second advantage of a CLEC is that cross-linking of the crystallizedenzyme stabilizes and strengthens the crystal lattice and theconstituent enzyme molecules, both mechanically and chemically. As aresult, a CLEC may be the only means of achieving significantconcentrations of active enzyme catalyst in harsh aqueous, organic,near-anhydrous solvents, or in aqueous-organic solvent mixtures. The useof enzymes as catalysts in organic syntheses has been hampered by theirtendency to denature in the presence of non-aqueous solvents, andparticularly, in mixtures of aqueous and non-aqueous solvents (Klibanov,A. M., Trends in Biochemical Sciences, 14:141-144 (1989)). In CLECs, therestriction of conformational mobility that leads to stability isprovided by the inter-molecular contacts and cross-links between theconstituent enzyme molecules making up the crystal lattice, rather thanby the near-absence of water in the medium. As a result, intermediatewater concentrations can be tolerated by enzymes when formulated asCLECs, as has previously not been possible (see Table 12). In commercialapplications, aqueous-organic solvent mixtures allow manipulation ofproduct formation by taking advantage of relative solubilities ofproducts and substrates. Even in aqueous media, enzyme catalysts,immobilized or soluble, are subject to mechanical forces within achemical reactor that can lead to denaturation and a shortenedhalf-life. The chemical cross-links within the CLEC provide thenecessary mechanical strength (Quiocho and Richards, Proc. Natl. Acad.Sci. (U.S.A.) 52: 833-839 (1964)) that results in increased reactor lifefor the enzyme catalyst.

A third advantage of a CLEC is that as a result of its crystallinenature, a CLEC can achieve uniformity across the entire cross-linkedcrystal volume. Crystalline enzymes as described herein are grown andcross-linked in an aqueous environment and, therefore, the arrangementof molecules within the crystal lattice remains uniform and regular.This uniformity is maintained by the intermolecular contacts andchemical cross-links between the enzyme molecules constituting thecrystal lattice, even when exchanged into other aqueous, organic ornear-anhydrous media, or mixed aqueous/organic solvents. In all of thesesolvents, the enzyme molecules maintain a uniform distance from eachother, forming well-defined stable pores within the CLECs thatfacilitate access of substrate to the enzyme catalysts, as well asremoval of product. Uniformity of enzyme activity is critical inindustrial, medical and analytical applications where reproducibilityand consistency are paramount.

A fourth advantage of using a CLEC is that it should exhibit anincreased operational and storage half-life. Lattice interactions, evenin the absence of cross-linking, are known to stabilize proteins, due inpart to restrictions of the conformational degrees of freedom needed forprotein denaturation. In CLECs, the lattice interactions, when fixed bychemical cross-links, are particularly important in preventingdenaturation, especially in mixtures of aqueous and non-aqueous solvents(Klibanov, A. M., Trends in Biochemical Sciences 14: 141-144 (1989)).Enzymes that have been in the crystalline state for months or yearsroutinely retain a high percentage of their catalytic activity.Cross-linked immobilized enzyme crystals stored in anhydrous solventswill be even further protected from microbial contamination and damage,which is a serious problem in storing large quantities of protein in anutrient rich, aqueous environment.

In the case of a lyophilized CLEC, the immobilized enzyme is stored inthe absence of solvent. That, and the stabilization achieved bycross-linking allows for the storage in the absence of refrigeration forlong periods of time.

A fifth advantage of using a CLEC is that it should exhibit enhancedtemperature stability as a consequence of the cross-links stabilizingthe crystal lattice. Carrying out reactions at a higher temperature thanthat used with conventional methods would increase reaction rates forthe chemical reactions of interest, both thermodynamically, and byenhancing the diffusion rate into and out of the crystal lattice ofCLECs. These combined effects would represent a major improvement inreaction efficiency, because they would maximize the productivity of agiven quantity of enzyme catalyst, which is generally the most expensivecomponent of the reaction process (Daniels, M. J., Methods in Enzymol.136: 371-379 (1987)). The temperature stability exhibited by CLECs isremarkable because most enzyme systems require mild reaction conditions.CLECs would also be stabilized against denaturation by transient hightemperatures during storage.

A final advantage of use of a CLEC is that pores of regular size andshape are created between individual enzyme molecules in the underlyingcrystal lattice. This restricted solvent accessibility greatly enhancesthe metal ion or cofactor retention characteristics of CLEC as comparedto conventionally immobilized enzymes and enzymes in solution. Thisproperty of CLEC will permit the use of economically superiorcontinuous-flow processes in situations (see e.g. Oyama et. al. Methodsin Enzymol. 136 503-516 (1987)) where enzyme would otherwise beinactivated by metal ion or cofactor leaching. For example, in thethermolysin-mediated synthesis of the dipeptidyl aspartame precursor,Z-L-Asp-L-Phe-OMe, conventionally immobilized enzyme is known to losecatalytic activity in continuous-flow column processes, in part throughthe leaching of calcium ions essential for thermolysin activity. Inpractice, leaching of calcium ions has forced the use of less efficientbatch processes (Nakanishi et. al., Biotechnology 3: 459-464 (1985)).Leaching occurs when calcium ion complexes are formed with substrateZ-L-Asp, in competition with the natural calcium binding sites on thesurface of the enzyme, resulting in the loss of catalytic activity. Thehigh density of enzyme, and the correspondingly limited volumeaccessible to solvent in the interstices of the CLECs, discourages theformation of the competing L-Asp-Ca⁺⁺ complexes responsible for metalion leaching.

In addition, crystallized, crosslinked antibodies, or CLACs, made by amethod similar to that used to produce CLECs, are the subject of thepresent invention. As described with reference to CLECs, althoughcrosslinking contributes to the stability of a crystallized antibody, itis neither necessary nor desirable in all instances. Crystallizedantibodies which are not subsequently crosslinked can be used in someembodiments of the present invention. CLACs of the present inventionhave the same advantages as described herein for CLECs. A particularlyuseful advantage is the enhanced resistance to degradation (e.g.,protease degradation) of CLACs.

Preparation of CLECs--Enzyme Crystallization

In the method of the present invention, a cross-linked immobilizedenzyme crystal (or CLEC) is prepared as follows:

Enzyme crystals are grown by the controlled precipitation of protein outof aqueous solution, or aqueous solution containing organic solvents.Conditions to be controlled include, for example, the rate ofevaporation of solvent, the presence of appropriate co-solutes andbuffers, and the pH and temperature. A comprehensive review of thevarious factors affecting the crystallization of proteins has beenpublished by McPherson (Methods Enzymol. 114: 112 (1985)). In addition,both McPherson and Gilliland (J. Crystal Growth 90: 51-59 (1988)) havecompiled comprehensive lists of all proteins and nucleic acids that havebeen reported as crystallized, as well as the conditions that lead totheir crystallization. A compendium of crystals and crystallizationrecipes, as well as a repository of coordinates of solved protein andnucleic acid crystal structures, is maintained by the Protein Data Bank(Bernstein et. al. J. Mol. Biol. 112: 535-542 (1977)) at the BrookhavenNational Laboratory. Such references can be used to determine theconditions necessary for the crystallization of a given protein orenzyme previously crystallized, as a prelude to the formation of anappropriate CLEC, and can guide the formulation of a crystallizationstrategy for proteins that have not. Alternatively, an intelligent trialand error search strategy (see eg., Carter, C. W. Jr. and Carter, C. W.,J. Biol. Chem. 254: 12219-12223 (1979)) can, in most instances, producesuitable crystallization conditions for most proteins, including, butnot limited to, those discussed above, provided that an acceptable levelof purity can been achieved for these. The level of purity required canvary widely from protein to protein. In the case of lysozyme, forexample, the enzyme has been crystallized directly from its unpurifiedsource, the hen egg-white (Gilliland, G. L., J. Crystal Growth 90: 51-59(1988)). For use as CLECs in the method of this invention, the largesingle crystals which are needed for X-ray diffraction analysis are notrequired, and may, in fact, be undesirable because of diffusion problemsrelated to crystal size. Microcrystalline showers (ie., crystals in theorder of 10⁻¹ mm in size/cross section) are suitable for CLECs and areoften observed, although seldom reported in the X-ray crystallographicliterature. Micro-crystals are very useful in the method of thisinvention to minimize problems with diffusion (see eg., Quiocho, F. A.,and Richards, F. M., Biochemistry 5: 4062-4076 (1967)).

In general, crystals are produced by combining the protein to becrystallized with an appropriate aqueous solvent or aqueous solventcontaining appropriate precipitating agents, such as salts or organics.The solvent is combined with the protein at a temperature determinedexperimentally to be appropriate for the induction of crystallizationand acceptable for the maintenance of protein stability and activity.The solvent can optionally include co-solutes, such as divalent cations,co-factors or chaotropes, as well as buffer species to control pH. Theneed for co-solutes and their concentrations are determinedexperimentally to facilitate crystallization. In an industrial scaleprocess, the controlled precipitation leading to crystallization canbest be carried out by the simple combination of protein, precipitant,co-solutes, and optionally buffers in a batch process. Alternativelaboratory crystallization methods, such as dialysis, or vapor diffusioncan also be adapted. McPherson (Methods Enzymol. 114: 112 (1985)), andGilliland (J. Crystal Growth 90: 51-59 (1988)) include a comprehensivelist of suitable conditions in their reviews of the crystallizationliterature. Occasionally, incompatibility between the cross-linkingreagent and the crystallization medium might require exchanging thecrystals into a more suitable solvent system.

Many of the proteins for which crystallization conditions have alreadybeen described in the literature, have considerable potential aspractical enzyme catalysts in industrial and laboratory chemicalprocesses, and are directly subject to formulation as CLECs within themethod of this invention. Table 1 is a sampling of enzymes that havealready been crystallized. Note that the conditions reported in most ofthese references have been optimized for the growth of large,diffraction quality crystals, often at great effort. Some degree ofadjustment of conditions for the smaller crystals used in making CLECSmight be necessary in some cases.

                                      TABLE 1                                     __________________________________________________________________________               Microbial or  References                                             Enzyme biological source (including those cited therein)                    __________________________________________________________________________    alcohol dehydrogenase                                                                    horse liver   Eklund et al., J. Mol. Biol. 146: 561-                   587 (1981)                                                                  alcohol oxidase Pichia pastoris Boys et al., J. Mol. Biol. 208: 211-212         (1989)                                                                        Tykarska et al., J. Protein Chem. 9:                                          83-86 (1990)                                                                aldolase rabbit muscle Eagles et al., J. Mol. Biol. 45: 533-544                                       (fructose-bisphosphale)  (1969)                         Heidmer et al., Science 171: 677-680                                          (1971)                                                                       calf muscle Goryunov et al., Biofizika 14: 1116-                               1117 (1969)                                                                  human muscle Millar et al., Trans. Roy. Soc. Lond.                             B293: 209-214 (1981)                                                         Drosophila melanogaster Brenner et al., J. Biol. Chem. 257:                    11747-11749 (1982)                                                          aldolse (PKDG) Pseudomonas puitda Vandlen et al., J. Biol. Chem. 248:                                   2251-2253 (1973)                                    alkaline phosphatase Escherichia coli Sowadski et al., J. Mol. Biol.                                 150:                                                     245-272 (1981)                                                              asparaginase Erwinia caratova North et al., Nature 224: 594-595                                         (1969)                                               Escherichia coli Epp et al., Eur. J. Biochem. 20: 432-                         437 (1971)                                                                   Escnerichia coli Yonei et al., J. Mol. Biol. 110: 179-                         186 (1977)                                                                   Proieus vulgaris Tetsuya et al., J. Biol. Chem. 248:                           7620-7621 (1972)                                                            carbonic anhydrase human erythrocyte (C) Kannen et al., J. Mol. Biol.                                12: 740-                                                 760 (1965)                                                                   human erythrocyte (B) Kannen et al., J. Mol. Biol. 63: 601-                    604 (1972)                                                                   bovine erythrocyte Carlsson et al., J. Mol. Biol. 80L 373:                     375 (1973)                                                                  catalase horse erythrocyte Glauser et al., Acta Cryst. 21: 175-                                         177 (1966)                                           Micrococcus luteus Marie et al., J. Mol. Biol. 129: 675-                       676 (1979)                                                                   Penicillium vitale Vainshtein et al., Acta Cryst. A37:                         C29 (1981)                                                                   bovine liver Eventoff et al., J. Mol. Biol. 103: 799-                          801 (1976)                                                                  creatine kinase bovine heart Gilliland et al., J. Mol. Biol. 170: 791-                                  793 (1983)                                           rabbit muscle McPherson, J. Mol. Biol. 81: 83-86                               (1973)                                                                      glutaminase Actenobacter glutanimasificans Wlodawer et al., J. Mol.                                  Biol. 99: 295-                                           299 (1975)                                                                   Pseudomonas 7A Wlodawer et al., J. Mol. Biol. 112:                             515-519 (1977)                                                              glucose oxidase Aspergillus niger Kalisz et al., J. Mol. Biol. 213:                                  207-                                                     209 (1990)                                                                  β-lactamases Staphylococcus aureus Moult et al., Biochem J. 225:                                167-                                                     176 (1985)                                                                   Bacillus cereus Sutton et al., Biochem J. 248: 181-                            188 (1987)                                                                  lacate dihydrogenase porcine Hackert et al., J. Mol. Biol. 78: 665-                                     673 (1973)                                           chicken Pickles et al., J. Mol. Biol. 9: 598-600                               (1964)                                                                       dogfish Adams et al., J. Mol. Biol. 41: 159-188                                (1969)                                                                       Bacillus stearothermophilus Scar et al., J. Mol. Biol. 154: 349-353                                    (1982)                                              lipase Geotrichum candidum Hata et al., J. Biochem. 86: 1821-1827                                       (1979)                                               horse pancreatic Lombardo et al., J. Mol. Biol. 205: 259-                      261 (1989)                                                                   Mucor meihei Brady et al., Nature 343: 767-770                                 (1990)                                                                       human pancreatic Winkler et al., Nature 343: 771-774                           (1990)                                                                      Luciferase Firefly Green, A. A., et al., Biochem. Biophys.                      Acta. 20: 170 (1956)                                                        luciferase Vibrio harveyii Swanson et al., J. Biol. Chem. 260:                  1287-1289 (1985)                                                            nitrile hydratase Brevibacterium R312 Nagasawa et al., Biochem.                                      Biophys. Res.                                            Commun. 139: 1305-1312 (1986)                                                P. chloroaphis B23 Nagasawa et al., Eur. J. Biochem. 162:                      691-698 (1987)                                                              peroxidase horseradish Braithwaite et al., J. Mol. Biol. 106:                   229-230 (1976)                                                               horshradish roots (Type E4) Aibara et al., J. Biochem. 90: 489-496                                     (1981)                                               Japanese radish Morita Acta Cryst. A28: S52 (1979)                           peroxidase (chloride) Caldaromyces fumago Rubin et al., J. Biol. Chem.                               257: 7768-                                               7769 (1982)                                                                 peroxidase (cytochrome) Sarchomyces cerevisae Poulos et al., J. Biol.                                Chem. 253:                                               3730-3735 (1978)                                                            peroxidase (glutathione) bovine erythrocyte Ladenstein et al., J. Mol.                               Biol. 104:                                               877-882 (1979)                                                              subtilisin Bacillus subtilis (Novo) Drenth et al., J. Mol. Biol. 28:                                 543-544                                                  (1967)                                                                       Bacillus amyloliquefaciens Wright et al., Nature 221: 235-242                 (BPN) (1969)                                                                  Bacillus subtilis (Carlsberg) Petsko et al., J. Mol. Biol. 106: 453-                                   456 (1976)                                          superoxide dismutase bovine Richardson et al., J. Biol. Chem. 247:                                      6368-6369 (1972)                                     spinach Morita et al., J. Biol. Chem. 86: 685-686                              (1974)                                                                       Saccharomyces cerevisiae, Beem et al., J. Biol. Chem. 105: 327-332                                    Escherichia coli (1976)                               Bacillus stearothermophillus Bridgen et al., J. Biol. Chem. 105: 333-                                  335 (1976)                                           Pseudomonas ovalis Yamakura et al., J. Biol. Chem. 251:                        4792-4793 (1976)                                                            thermolysin Bacillus thermoproteolyticus Matthews et al., Nature New                                 Biol.                                                    238: 37-41 (1972)                                                           urease jack bean Sumner, J. B., J. Biol. Chem. 69: 435                          (1926)                                                                      xylose isomerase Streptomyces rubiginosus Carrell et al., J. Biol.                                   Chem. 259:                                               3230-3236 (1984)                                                             Arthrobacter B3728 Akins et al., Biochym. Biophys Acta                         874: 375-377 (1986)                                                          Streptomyces olivochromogenes Farber et al., Protein Engineering 1:                                    459-466 (1987)                                       Streptomyces violaceoniger Glasfeld et al., J. Biol. Chem. 263:                                        14612-14613 (1988)                                   Actinoplanes missouriensis Rey et al., Proteins: Struc. Func. Genet.                                   4: 165-172 (1988)                                 __________________________________________________________________________

Preparation of CLECs--Cross-Linking Reaction

Once crystals are grown in a suitable medium, they can be cross-linked.Cross-linking results in stabilization of the crystal lattice byintroducing covalent links between the constituent enzyme molecules inthe crystal. This makes possible the transfer of enzyme into analternate reaction environment that might otherwise be incompatible withthe existence of the crystal lattice, or even with the existence ofintact undenatured protein. Cross-linking can be achieved by a widevariety of bifunctional reagents, although in practice, simple,inexpensive glutaraldehyde has become the reagent of choice. (For arepresentative listing of other available cross-linking reagents, onecan consult, for example, the 1990 catalog of the Pierce ChemicalCompany). Cross-linking with glutaraldehyde forms strong covalent bondsbetween primarily lysine amino acid residues within and between theenzyme molecules in the crystal lattice that constitute the crystal. Thecross-linking interactions prevent the constituent enzyme molecules inthe crystal from going back into solution, effectively insolubilizing orimmobilizing the enzyme molecules into microcrystalline (ideally 10⁻¹mm) particles. The macroscopic, immobilized, insolubilized crystals canthen be readily separated from the feedstock containing product andunreacted substrate by simple procedures such as filtration,decantation, and others. They can also be used in CLEC packed columns incontinuous flow processes, where they exhibit enhanced cofactor andmetal ion retention properties.

By the method of this invention, CLECs are obtained for use as enzymecatalysts in existing and novel environments. The enhanced stability ofthe CLECs, which results from the cross-linking reaction, makes itpossible to transfer the CLEC into a solvent (e.g., aqueous, organic ornear-anhydrous solvents, or a mixture of these), in which it wouldotherwise be incompatible, and to carry out chemical reactor operationat elevated temperatures of extremes of pH. The macroscopic CLECcatalyst particles can also be readily manipulated, allowing recoveryfrom feedstock by simple methods, such as filtration, centrifugation, ordecantation of solvent. In addition, these can be used in packed columnsin continuous flow processes.

Preparation of CLECs--Lyophilization

A suspension of one volume of cross-linked thermolysin crystals in tenvolumes of demineralized water at pH 7.0 was lyophilized overnight usinga VirTis Model #24 lyophilizer. Lyophilized crystals were stored at roomtemperature or at 4° C. prior to reconstitution, which was accomplishedby adding ten volumes of the solvent of choice directly onto crystalstaken from storage. Re-hydrated crystals were reconstituted in 10 mMcalcium acetate buffer at pH 7.0 for the FAGLA cleavage experiments.Reconstituted lyophilized CLECs were routinely stored at roomtemperature. In contrast, soluble enzyme required storage at -70° C. tomaintain specific activity longer than a week. This protocol was usedfor all the enzymes described in the exemplification included here.

Synthesis of Aspartame Precursor With Thermolysin CLEC

The method of the present invention, by which cross-linked crystalenzymes are produced, is described below and exemplified by theproduction of cross-linked immobilized enzyme crystals of thermolysinfor use in the production of the dipeptidyl precursor of aspartame, inethyl acetate, which is a near-anhydrous organic, solvent. Thermolysin,a protein which has been crystallized and whose structure has beensolved at 1.6 Å resolution (Holmes and Matthews, J. Mol. Biol. 160:623-639 (1982)), is one example of an enzyme which can be used as a CLECin the present method. Thermolysin is used in the manufacture of theartificial sweetener aspartame (Isowa et. al. U.S. Pat. No. 4,436,925(1984); Lindeberg, J. Chem. Ed. 64: 1062-1064 (1987); Nakanishi et. al.,Biotechnology 3: 459-464 (1985); Oyama, et. al., Methods in Enzymol.136: 503-516 (1987)). At the present time, most aspartame appears to beproduced by a conventional synthetic chemistry approach, although use ofconventionally immobilized thermolysin in near-anhydrous media hasproduced encouraging results (Oyama et. al., J. Org. Chem. 46: 5242-5244(1981); Nakanishi et. al., Biotechnology 3: 459-464 (1985)). Improvementin the enzymatic approach to aspartame production, such as is possiblethrough use of the present method, would make it competitive with thepresently-used method, both in terms of convenience and cost (Oyama, et.al., Methods in Enzymol. 136: 503-516 (1987)).

Assessment of Thermolysin CLECs

The method of the present invention has also been used to producethermolysin CLECs which have been assessed as to their pH dependence andstability, stability at elevated temperature, resistance to exogenousproteolysis and stability in the presence of an organic solvent.Thermolysin CLECs were compared to soluble thermolysin, as described indetail in Example 2 and FIGS. 1-4. Results of the assessment showed thefollowing:

1. As to pH dependence and stability, both forms demonstrate maximumactivity at pH 7 and demonstrate similar activity in the acidic range.In the alkaline pH range, the CLEC maintains maximum activity to pH 10;the soluble thermolysin has 75% activity at pH 8.5, only 25% activity atpH 9 and is completely inactive at pH 9.5.

2. The additional stabilization achieved in CLECs results in enzymaticactivity at higher temperatures than is possible with solublethermolysin. Enhanced stability of CLEC thermolysin at lowertemperatures makes storage simpler than it is for the soluble enzyme.Thermal stability and resistance to autolysis was also demonstrated forthermolysin CLECs, which retained maximum activity after five days ofincubation at 65° C. In contrast, soluble thermolysin lost 50% of itsinitial activity after two hours incubation and demonstrated negligibleactivity after 24 hours incubation at 65° C.

3. Enzymatic activity of thermolysin CLECs was unaffected by four days'incubation in the presence of the powerful streptococcal protease,Pronase®. In contrast, soluble thermolysin was rapidly degraded and lostall activity after 90 minutes incubation.

4. Thermolysin CLECs and soluble thermolysin exhibited markedlydifferent stability in the presence of organic solvents, as shown inTable 12. The thermolysin CLECs retained greater than 95% maximumactivity following incubation with all organic solvents assessed.Additional work with soluble and CLEC thermolysin catalysed synthesis isdescribed in Example 6 and FIG. 5.

These features of thermolysin CLECs and other enzyme CLECs make themparticularly useful, since they are easier to store, more stable andless easily inactivated or degraded than corresponding soluble enzymes.

Assessment of Elastase CLECs

The method of the present invention has also been used to produceelastase CLECs which have been assessed as to their activity andresistance to exogenous proteolysis. Elastase CLECs were compared tosoluble elastase, as described in detail in Example 4 and FIGS. 6 and 7.Results of the assessment demonstrated the following:

1. Elastase CLECs retain approximately 50% activity compared to solubleenzyme.

2. Soluble elastase was rapidly degraded by protease. Activity ofsoluble elastase was reduced to 50% of initial activity following tenminutes incubation in the presence of protease. After one hourincubation the soluble enzyme had lost more than 90% of its initialactivity. In contrast the enzymatic activity of the elastase CLEC wasunaffected by incubation with protease.

Assessment of Esterase CLECs

The method of the present invention has also been used to produceesterase CLECs which have been assessed as to their activity andresistance to exogenous proteolysis. Esterase CLECs were compared tosoluble esterase, as described in detail in Example 5 and FIGS. 8 and 9.Results of the assessment demonstrated the following:

1. Esterase CLECs retain approximately 50% activity compared to solubleenzyme.

2. Soluble esterase was highly susceptible to proteolytic degradation.Activity of soluble esterase was reduced to 50% of initial activityfollowing ten minutes incubation in the presence of protease. After onehour incubation the soluble enzyme had lost more than 90% of its initialactivity. In contrast the enzymatic activity of the esterase CLEC wasunaffected by incubation with protease.

Assessment of Lipase CLECs

The method of the present invention has also been used to produce lipaseCLECs which have been assessed as to their activity. Lipase CLECs werecompared to soluble lipase, as described in detail in Examples 6 and 10and FIG. 9. Results of the assessment demonstrated that G. candidum andC. cylindracea lipase CLECs retain significant activity, compared tosoluble enzyme (Examples 6 and 10) and that the porcine pancreaticlipase retained activity to a limited extent. (Example 11)

Assessment of Lysozyme CLECs

The method of the present invention has also been used to producelysozyme CLECs which have been assessed as to their activity andresistance to exogenous proteolysis. Lysozyme CLECs were compared tosoluble lysozyme as described in detail in Example 7 and FIG. 11.Results of the assessment demonstrated that lysozyme CLECs retainapproximately 50% activity compared to soluble enzyme.

Assessment of Asparaginase CLECs

The method of the present invention has also been used to produceasparaginase CLECs which have been assessed as to their activity.Asparaginase CLECs were compared to soluble asparaginase, as describedin detail in Example 8 and FIG. 12. Results of the assessmentdemonstrated the following asparaginase CLECs retain approximately 77%activity compared to soluble enzyme.

Assessment of Urease CLECs

The method of the present invention has also been used to produce ureaseCLECs which have been assessed as to their activity. Urease CLECs werecompared to soluble urease, as described in detail in Example 9 andFIGS. 13-16 and, in addition, urease CLEC activity in an aqueous bufferwas compared with its activity in sera, which is a biologically relevantmedium. (Example 9 and FIG. 17) Results of the assessment demonstratedthat urease CLECs retain significant enzymatic activity and that therewas comparable urease activity in aqueous buffer and in sera.

General applicability of CLECs

As disclosed here, CLECs represent a novel technology with broad use inmany areas, including, but not limited to, industrial scale syntheses,laboratory tools, biosensors, and medical applications. Examples ofvarious systems using conventionally immobilized enzyme methods in theirexecution are given in Tables 2-5 below. One skilled in the art shouldbe able to adapt these, and similar systems, to the CLEC technologydisclosed in this application. To illustrate this, specific examples arediscussed in more detail from each of the categories listed.

Table 2 below lists examples which use conventionally immobilizedenzymes in an industrial process, which examples can be readily adaptedto the CLEC technology disclosed here.

                                      TABLE 2                                     __________________________________________________________________________               Production or         References                                     Enzyme application Substrates (including those cited)                       __________________________________________________________________________    thermolysin                                                                              aspartame precursor                                                                     Z-Asp, L-Phe-OMe                                                                          Oyama et al J. Org. Chem.                         46: 5242-5244 (1981)                                                          Nakanishi et al., Trends in                                                   Biotechnology 3: 459-464                                                      (1985)                                                                     subtilisin aspartame L-Asp-L-Phe, OMe Divino, A. A., U.S. Pat. No.                                               4,293,648 (1981)                           lipase cocoa fat palm oils Harwood, J., Trends in                              substitutes  Biochemical Sciences 14: 125-                                      126 (1989)                                                                    Macrae, A. R., J. Am. Oil                                                     Chem Soc. 60: 291-294                                                         (1983)                                                                     mitrile hydratase, acrylamide acrylonitrile Nagasawa, T. and Yamada,                                         H.,                                            nitrilases, amidase   Trends in Biotechnology 7:                                 153-158 (1989)                                                             amino acylase (fungal) amino acid N-acryl-D,L amino acids Schmidt-Kastne                                     r, G. & Egerer,                                amino acid esterase, resolution esters of D,L amino P. in Biotechnology                                      vol 6a:                                        subtilisin  acids 387-421 (1984) and references                               amidases amides of D,L amino therein.                                         hydantionases  acids Fusse, M. C., Methods in                                 specific dehydrogenases  hydantoins Enzymology 136: 463 (1987)                amino peptidase  a-hydroxycarboxylic Fusse, M. C., Methods in                 transaminase  acids Enzymology 136: 479 (1987)                                amino acid amino acid keto or hydroxy acids Rozzell, J. D., Methods in                                        dehydrogenase + formate production:                                          general  Enzymology 136: 479 (1987)                                            dehydrogenase amino acid Enyzmes in                                          Industry; Ed                                    production: specific  Gerhartz. W., VCH Press 1990                           L-aspartase L aspartic acid fumarate/fumaric acid                             L-aspartate 4- L-alanine L-aspartic acid,                                     decarboxylase  ammonium fumarate                                              aspartase + L aspartate 4                                                     decar-boxylase L-lysine D,L-a amino e-                                        ACL hydrolase  caprolactam (ACL)                                              L-ACT hydrolase L-cysteine DL-2amino2 thiazoline                                4carboxylic acid                                                             L-isoleucine                                                                  L-methionine                                                                 lyase L-phenylalanine cinnamate                                               L-tryptophan synthetase L-tryptophan indole, L-serine                          L-valine                                                                     fumarase L-malic acid fumarate                                                hydantoinase D n carbamoyl p- 5p-hydroxy hydantoin                             hydroxy- phenyl                                                               glycine                                                                      lipases, esterases resolution of synthetic chemistry Jones, J. B.,                                           Tetrahedron 42:                                 racemates by  3351-3403 (1988)                                                stereoselective  Butt, S. and Roberts, S. M.,                                 synthesis  Natural Product Reports 489-                                         503 (1986), and references cited                                              therein for a more                                                            comprehensive review of this                                                  area                                                                       fumarase L-malic acid fumaric acid Chibata et al., Methods in                    Enzymology 136: 455 (1987)                                                 lactase, β-galactosidases desaccharide lactose & N-acetyl Larsson                                       et al., Methods in                              synthesis eg galactosamine Enzymology 136: 230 (1987)                         galactosyl-N-acetyl                                                           galactosamine                                                                lipase, esterase L-menthol 4 isomer mix Fukui, S., Tanaka, A., Methods                                           in Enzymology 136: 293                        (1987)                                                                     amidases D-valine D,L amino acid amide Schmidt-Kastner, G. & Egerer,                                           (intermediate for  P. in Biotechnology                                      vol 6a:                                         pyrethroid  387-421 (1984) and references                                     insecticide fluvinate)  therein.                                             lipase (Candida R(+)2 phenoxy- 2 chloro propionic acids Biocatalysts in                                      Organic                                        cylindricea) propionic acids  Syntheses eds Tramper, van de                    (herbicides)  Plas & Linko: Proceedings of                                      International Symposium in                                                    Netherlands 1985                                                           lipases, esterases, organic syntheses  Jones, J. B., Tetrahedron 42:                                          amidases, aldolases monoglycerides                                           3351-3403 (1988)                                peptides  Butt, S. and Roberts, S. M.,                                       proteases, peptidases   Natural Product Reports 489-                             503 (1986), and references cited                                           yeast lipase 2(p-chlorophenoxy) resolution of racemic therein for a                                          more                                            propionic acid: ester comprehensive review of this                            herbicide  area                                                              strictodine synthetase alkaloid production  Pfitzner et al., Methods in        eg strictosidine  Enzymology 136: 342 (1987)                                 penicillin acylase 6-amino penicillin G or V Enz Eng 6: 291 (1982)                                            penicillin amidase penicillinanic acid                                       Enz Eng 8: 155                                  and 7-ADCA                                                                   hydroxysteroid steroid  Carrea et al., Methods in                             dehydrogenases transformations  Enzymology 136: 150 (1987)                    5'phosphodiesterase, 5'-ribonucleotides  Keller et al., Methods in                                            nucleases   Enzymology 136: 517 (1987)                                        esterase β-lactam precursor                                             corresponding diesters Japanese patent                                        application:                                    (chiral mono esters  82-159, 493 (1981) Biseibutsu                            eg βamino glutaric  Company                                              acid monoalkyl                                                                ester)                                                                       lipases β-blockers  Kloosterman, M. et al., Trends in                       Biotechnology 6: 251-256                                                      (1988)                                                                   __________________________________________________________________________

Production of Acrylamide Using CLEC Technology

The following is a description of one use of the method of the presentinvention: the adaptation of acrylamide production from immobilizedcells which overproduce nitrile hydratase enzyme (Nagasawa, T. andYamada, H., Trends in Biotechnology 7: 153-158 (1989)) to the CLECtechnology previously disclosed herein.

Industrial scale production of acrylamide, an important commoditychemical, has been described by Yamada and collaborators (Nagasawa, T.and Yamada, H., Trends in Biotechnology 7: 153-158 (1989)). Kilotons ofacrylamide per year are produced in chemical reactors loaded withentrapped cells selected as overproducers of the enzyme nitrilehydratase. Nitrile hydratase has also been reported as purified andcrystallized from two sources, Brevibacterium R312 (Nagasawa et. al.,Biochem. Biophys. Res. Commun. 139: 1305-1312 (1986) and P. chlororaphisB23 (Nagasawa et. al., Eur. J. Biochem. 162: 691-698 (1987). Asdisclosed here, these crystalline enzymes can each be immobilized bycrosslinking with glutaraldehyde or other suitable crosslinking reagentto produce a CLEC. The nitrile hydratase CLECs can then be used in aconventional reactor, in place of the entrapped cells currently used.Adapting this process to CLEC technology leads to immediate advantages.These include; reduced plant size and improved throughput resulting fromthe enhanced activity per unit volume implicit in the higher enzymeconcentration in the CLECs, and improved substrate and product diffusionrates; reduction in undesired contamination and side reactions,resulting from the higher purity of CLECS; and reduced sensitivity tomicrobial contamination in the absence of cells. In addition, there areother benefits available only to a CLEC-based method. These benefitsinclude: higher temperature operation to improve reaction rates; theability to operate in aqueous, organic and near-anhydrous solvents,allowing optimization of the acrylamide production reaction; andenhanced half-life in operation and storage, resulting from the higherchemical and mechanical stability of CLECS, particularly inunconventional solvents.

Medical Applications of CLEC Technology

The method of the present invention and an appropriately selected CLECor set of CLECs can also be used for medical applications. A CLEC or aset of CLECs can be used, for example, to remove a component of a fluid,such as blood, generally by altering the component, and thus, convertingit to a substance not detrimental to an individual or which can beremoved by normal body processes (e.g., via detoxification, ordegradation in the liver, excretion via the kidneys). In thisapplication, an appropriately-selected CLEC or set of CLECs is broughtinto contact with body fluid, which contains the component to bealtered, or a reactant (product or substrate) of a reaction in which thecomponent participates, upon which the enzyme in the CLEC acts. As aresult, the enzyme is able to act upon the component to be altered orwith another substance which is a product of a reaction in which thecomponent to be altered participates. The activity of the enzyme resultsin direct alteration of the component to be removed or in alteration ofthe product of the reaction in which the component participates (thusmaking continuation of the reaction impossible). This can be carried outthrough the use of an extracorporeal device which includes anappropriately-selected CLEC or set of CLECs and a retaining means whichis made of a material; such as a porous material on which a CLEC isretained or a tube in which a CLEC is present, which allows contactbetween the component itself or the substance in the fluid which is aproduct of a reaction in which the component to be altered participates.

This might also be achieved by the insertion of an appropriate CLEC intoa suitable body compartment, such as the peritoneum or a lymph node,where the CLEC would have access to bodily fluids. This insertion mightbe done surgically, or by injection of the CLEC slurry. Direct injectionof CLEC into the blood stream would not be appropriate, given the highrisk of embolism.

The use of appropriate CLECs in this area might serve as an alternativeto genetic methods in enzyme replacement therapy to correct a naturaldeficiency, such as, e.g. phenylketonuria.

Table 3 illustrates some of the medical applications in which CLECscould be used. For the majority of these cases, the extra-corporealtreatment is still in the research phase, but the benefits that CLECsoffer could provide novel treatments in areas in which there waspreviously no alternative treatment.

                                      TABLE 3                                     __________________________________________________________________________    Enzyme                                                                          employed Removal of:- Disease/patients treated References                   __________________________________________________________________________    asparaginase                                                                           asparagine                                                                             leukemia       Klein, M., Langer, R., Trends in                                                 (Removal of asparagine, an Biotechnolo                                     gy 4: 179-185                                    important cancer nutrient, harms (1986) and references therein                                                leukemic cells which cannot Chang, T.                                      M. S., Methods in                                manufacture the essential amino Enzymology 137: 444-457                       acid - asparagine; normal cells (1987) & references therein                   can manufacture asparagine and so                                             are unaffected by this treatment.)                                          heparinase heparin deheparinization for Langer, R., et al., Science                                          217:                                             hemoperfusion patients eg kidney 261-263 (1982)                               dialysis                                                                    bilirubin oxidase bilirubin neo-natal jaundice Lavin, A., et al.,                                            Science 230:                                     543-545 (1985)                                                              carboxypeptidase methotrexate chemotherapy patients Pitt, A. M., et                                          al., Appl.                                        Biochem. Biotechnol. 8: 55-68                                                 (1983)                                                                     tyrosinase aromatic amino liver failure exhibiting Chang, T. M. S.,                                          Sem. Liver                                      acids pathological elevations of amino Dis. Ser. 6: 148 (1986)                                                 acids                                       phenylalanine phenylalanine phenylketonuria and liver failure Ambrus,                                        C. M., et al.,                                 ammonium lyase   J. Pharm. & Exp. Ther. 224:                                     598-602 (1983)                                                             multi-enzyme urea (converted detoxification for chronic renal Chang, T.                                      M. S., Methods in                              system including: into glutamic and failure patients Enzymology 137:                                         444-457                                        urease, glutamate other amino acids,  (1987) & references therein                                             dehydro-genase, via ammonia)  Chang, T.                                      M. S., Enzyme Eng                              glucose,   5: 225 (1980)                                                      dehydrogenase & a                                                             transaminase                                                                  arginase arginine familial hyperargininaemia Kanalas, J. J., et al.,                                         Biochem.                                          Med. 27: 46-55 (1982)                                                      glutamate ammonia liver failure Maugh, T. H., Science 223:                    dehydrogenase &   474-476 (1984)                                              ammonia                                                                     __________________________________________________________________________

A particular application of the present method is the heparin lyasesystem for blood deheparinization (Bernstein et al., Methods inEnzymology 137: 515-529 (1987)), which is discussed below.

All extracorporeal devices perfused with blood, such as kidney dialysis,continuous arteriovenous hemofiltration or extracorporeal membraneoxygenators, require heparinization of the patient to avoid bloodclotting. However, heparinization of the patient leads to hemorrhagiccomplications and remains a threat to human safety. These problemsincrease as the perfusion times increase, for example with the membraneoxygenator, and can lead to serious bleeding. After extracorporealtherapy, heparin may be removed from blood by employing a heparinasedevice at the effluent of the extracorporeal device which eliminates allheparin from the blood returned to the patient and thus avoids thecurrent problems of heparinization.

Published research (Langer et al. Science 217: 261-263 (1982));Bernstein et al., Methods in Enzymology 137: 515-529 (1987)), detailsthe problems presented by conventionally immobilized enzymes used inextracorporeal devices. The principal problem is that conventionalimmobilization results in a low retention of enzyme activity per unitvolume, thus requiring a large volume of immobilized enzyme to performthe necessary heparinization. This volume is too large to be ofpractical use in humans. However, the high retention of activity perunit volume in CLECs, due to the lack of inert support circumvents thisproblem and offers a practical solution to deheparinization of humans.The enhanced stability of CLECs will reduce the disassociation of enzymefrom the crosslinked crystal. This is superior to the less stable,conventionally immobilized enzymes, because immune responses resultingfrom enzyme leakage will be reduced. CLEC temperature stability preventsdenaturation of the enzyme due to high transient temperatures duringstorage; it is likely that CLECs may retain high activity, even whenstored at room temperature. In addition, CLECs will be cheaper and moreconvenient to use than their conventionally immobilized counterpartsbecause of their longer operational and storage lifetimes.

CLECs made by the present method can be used for additional medicalpurposes, both therapeutic and diagnostic. As described herein, enzymeswhich have potential for such uses have been crystallized andcrosslinked and assessed as to their enzymatic activity and stabilityunder various conditions. For example, lipase CLECs have been producedand shown to retain significant enzymatic activity. Such lipase CLECscan be used, for example, to treat individuals with pancreaticinsufficiency and/or fat malasorption conditions, in which lipasesecretion is abnormally low. This can be associated with steatorrhea,essential fatty acid deficiency, loss of a high calorie source (fat) ora fat-soluble vitamin deficiency. Presently available approaches tolipase supplementation have numerous shortcomings, which limit theireffectiveness. For example, gastric acid inactivation of enzymesupplements or digestion by proteases of lipase-supplementation agentsmay occur, reducing the available amount of the agent(s) used. Possiblesupplementation strategies include high doses of pancreatic enzymes, useof pH sensitive enteric-coated microspheres and capsules, gastric acidmodulation and use of acid resistant lipases (which presently areunavailable). Lipase CLECs can be used as therapeutic agents or drugsand, in this context, have several key advantages: they are stable toexogenous proteolysis, stable to pH levels which would inactivate ordestroy other enzyme forms, stable to heat and solvents and easilystored because they can be lyophilized; as described herein. Othertherapeutic uses include administration of or treatment with urease.CLECs and xanthine oxidase CLECs (e.g., in treating hyperuriocosuria,resulting in conversion, in the GI tract, of purines to uric acid,followed by excretion of the uric acid.

In therapeutic applications, design and use of CLECs which release theenzyme over time (e.g., slow or controlled release) might proveadvantageous, such as to provide the activity over time or to delay itsrelease (e.g., to allow the enzyme to pass through harsh pH conditionsin the stomach by being protected in CLEC form and then being released).Production of CLECs in which crosslinking and/or crystallization isdesigned to permit slow or controlled release would provide usefulagents.

Additional Applications of CLEC Technology: Biosensors

A CLEC or a set of CLECs can be used as a component of a sensor,referred to as a biosensor, useful for detecting and/or quantitating ananalyte of interest in a fluid, such as body fluid (e.g., blood, urine),chemical and laboratory reaction media, organic media, water, culturemedium and beverages. In some instances, the fluid in question can be agas, as in an alcohol breath analyzer (Barzana, E., Klibanov, A., andKarell, M., NASA Tech Briefs 13:104 (1989)). In this application anappropriately-selected CLEC or set of CLECs is brought into contact witha fluid to be analyzed for the analyte of interest. The analyte ofinterest can be measured directly (e.g., blood glucose level) orindirectly (e.g., by detecting or quantitating a substance which is areactant (product or substrate) in a reaction in which the analyte ofinterest participates). In either case, the CLEC is able to act upon theanalyte or the substance which is a reactant in a reaction in which theanalyte also participates. The activity of the enzyme results in adetectable change (e.g., change in pH, production of light, heat, changein electrical potential) which is detected and/or quantitated by anappropriate detecting means (e.g., pH electrode, light or heat sensingdevice, means for measuring electrical charge) (Janata, J., et al.,Anal. Chem. 62: 33R-44R (1990)). Any means useful for detecting thechange resulting from the enzyme-catalyzed method can be used. Abiosensor of the present invention includes a CLEC or set of CLECs and aretaining means for the CLEC which allows contact between the CLEC(s)and the analyte of interest or the substance in the fluid which is areactant in the reaction in which the analyte of interest participates.

Table 4 illustrates some of the biosensor applications in which CLECscould be used. Currently immobilized enzymes are used in theseapplications, but suffer from low stability, low enzyme density, shortlifetimes and lack of reproducibility. These examples can be readilyadapted to the CLEC technology disclosed here.

                                      TABLE 4                                     __________________________________________________________________________    Enzyme Employed                                                                          Detection of:-                                                                           Application                                                                              Reference                                    __________________________________________________________________________    glucose oxidase                                                                          glucose    diabetics  Daniles, B., Mossbach, K.,                        Methods in Enzymology                                                         137: 4-7 (1987)                                                               Hall, E. "Biosensors" Open                                                    University Press (1990)                                                       Taylor, R., Proceed,                                                          Biotechnology Conference                                                      1989; 275-287                                                                 Anthony et al.,                                                               "Biosensors, Fundamentals                                                     and Applications", Oxford                                                     University Press (1987)                                                    creatinine deiminase creatinine kidney function Tabata, M. et al.,                                           Anal.                                             Biochem. 134: 44 (1983)                                                    urease urea kidney function Hsuie, G. H. et al.,                                 Polym. Mater. Sci. Eng. 57:                                                   825-829 (1987)                                                                Kobos, et al. Anal. Chem.                                                     60: 1996-1998 (1988)                                                       lactate oxidase & lactate clinical applications Blaedel, W. J. &                                             Jenkins, R.                                    dehydrogenase   A., Anal. Chem. 48(8):                                           1240 (1976)                                                                   Sagaguchi, Y., et al., J.                                                     Appl. Biochem 3: 32 (1981)                                                 glucose-6-pyruvate Glucose-6-phosphate, diabetics and other ibid as                                          glucose oxidase                                dehydrogenase sucrose and ATP medical                                         alcohol dehydrogenase, ethanol & other breathalysers and Romette, J. L.                                      et al.,                                        alcolol oxidase alcohols; acetic, formic industrial applications                                             Methods in Enyzmology                           acids  137: 217-225 (1987)                                                      Ho, M. H., Methods in                                                         Enzymology 137: 271-288                                                       (1987)                                                                     β-fructosidase sucrose industrial applications Romette, J. L. et                                        al.,                                              Methods in Enzymology                                                         137: 217-225 (1987)                                                        chloresterol oxidase cholesterol cholesterol testing Satoh, I., Methods                                      in                                                Enzymology 137: 217-225                                                       (1987)                                                                     catalase uric acid, cholesterol atherosclerotic and Satoh, I., Methods                                       in                                               other medical Enzymology 137: 217-225                                          (1987)                                                                     carboxy peptidase methotrexate cancer ibid as glucose oxidase                 carbonic anhydrase carbon dioxide industrial, laboratory & ibid as                                           glucose oxidase                                  environmental                                                                 applications                                                                L-amino acid oxidase amino acids medical and industrial ibid as glucose                                      oxidase                                        β-lactamase penicillin medical Anzai et al., Bull. Chem.                 penicillinase   Soc. Jpn. 60: 4133-4137                                          (1988)                                                                     alkaline phosphatase phosphate metabolic monitoring ibid as glucose                                          oxidase                                        nitrate/nitrite reductase nitrates & nitrites metabolite and food ibid                                       as glucose oxidase                               monitoring                                                                  arylsulfatase sulfate metabolite monitoring ibid as glucose oxidase                                           succinate succinate industrial ibid as                                       glucose oxidase                                dehydrogenase                                                                 bacterial luciferase FMNH.sub.2 and coupled detection of 10.sup.-18                                          Wannlund J., et al.,                            reactions molar quantities of "Luminescent assays:                             FMNH.sub.2 through Perspectives in                                            measurement of photon endocrinology and clinical                              release chemistry"; Eds Serio, M.                                              and Pazzagli, M. 1: 125                                                       (1982)                                                                        Kurkijarvi et al., Methods                                                    in Enzymology 137: 171-                                                       181 (1987)                                                                 firefly luciferase ATP and coupled detection of 10.sup.-12 Kurkijarvi                                        et al., Methods                                 reactions molar quantities of ATP in Enzymology 137: 171-                      through measurement of 181 (1987)                                             photon release Murachi et al., Methods in                                      Enzymology 137: 260-271                                                       (1988)                                                                   __________________________________________________________________________

In the method of the present invention as it is carried out for theanalysis of samples in a biosensor, it is particularly desirable toproduce the largest possible detectable signal from the smallestpossible quantity of substrate and catalyst. In this regard, the CLECtechnology disclosed here is particularly attractive, since it achievesthe highest possible concentration of enzyme catalyst in a given volume.

Often, considerable efforts are made to couple an ultimate enzymaticreaction of interest, either directly, or through suitableintermediates, to the production of light by enzymes like luciferase(Kurkijarvi et al., Methods in Enzymol. 137: 171-181 (1988)). This isdone in order to take advantage of the unparalleled sensitivity andefficiency of photon detection equipment, which allows for the detectionof femtomolar concentrations of enzyme reaction products underappropriate conditions. Following this principle, biosensor systems havebeen designed, using conventionally immobilized enzymes, to detectvarious substrates of clinical and other interest. Light producingreactions have been coupled to assay reactions detecting substrateslike, D-glucose, L-lactate, L-glutamate and ethanol, among others, atextremely low concentration.

With regard to this application, the luciferase enzyme from Vibrioharveyii has been reported as crystallized (Swanson et al., J. Biol.Chem. 260: 1287-1289 (1985)). Crystals of this luciferase can becrosslinked with glutaraldehyde or other suitable reagent to form a CLECof luciferase. For biosensor and analytical uses, a CLEC of luciferaseoffers many advantages over conventionally immobilized enzyme. In aCLEC, the entire volume of the luciferase CLEC would consist of lightemitting enzyme. In a conventionally immobilized enzyme system, however,as much as 95% of the total volume is taken up by "inert" carriermaterial, which more likely functions as an absorber of the lightemitted by enzyme. In addition, the enhanced stability of CLECs shouldfacilitate storage at room temperature, and also makes possible novelsensing applications in harsh environments and elevated temperatures.

CLACs of the present invention should be useful for diagnostic andtherapeutic purposes (e.g., delivery of an agent (such as a label or acytotoxic agent) to a defined cell type (one recognized by theantibody).

Additional Applications of CLEC Technology--Laboratory Reactions

CLECs may be used as laboratory reagents in small columns or in batchprocesses, which can be used to carry out laboratory reactions. Some ofthe broad categories of reactions are in Table 5. In addition,appropriately selected antibodies, or antibody fragments (particularlymonoclonal antibodies), which recognize process chemicals, clinicalanalytes, pesticides and other environmental residues can be turned intoCLACs and used to selectively bind these substances, making theirdetection in and/or removal from a sample or other source possible. Forexample, a substance can be separated from a mixture by contacting themixture with a crosslinked immobilized antibody crystal in which theantibody recognizes the substance, thereby producing a complex of thesubstance and the crystal, and separating from the mixture the resultingcomplex. The crosslinked crystal will generally be linked to a solidsupport, such as a column or a bead. For example, CLACs can be packedinto an affinity column and a sample from which a selected component isto be removed can be run through the column resulting in binding of thecomponent to the antibody and permitting its removal. This can be used,for example, on a small or a large scale to remove pesticides (e.g.,Aldrin) from soil, water or other source. In this case, a pesticidebinding monoclonal antibody CLAC is used in an affinity column or otherappropriate structure. Because it is in CLAC form, the monoclonalantibody can withstand the harsh conditions used (e.g., organicsolvents) and the antibody-bound pesticide is removed from the sample(e.g., by nature of the fact it is bound to antibody which is, itself,bound to a solid support, which can be separated from the sample.

                                      TABLE 5                                     __________________________________________________________________________    Enzyme Employed                                                                           Reaction type catalyzed                                                                       Reference                                         __________________________________________________________________________    lipases, phospholipases                                                                   Stereoselective synthesis: including                                                          Zaks, A. & Klibanov, A. M.                           esterification, transesterification, Proc. Nat. Acad. Sci. USA. 82:                                    3192-                                                aminolysis, lactonizations, 3196 (1985)                                       polycondensations, acylation, Klibanov, A. M. Acc. Chem. Res. 23:                                        oximolysis and resolution of racemic                                        114-120 (1990) and references therein                                           mixtures Wong, C. H., Chemtracts-Organic                                       Chemistry 3: 91-111 (1990) and                     references therein                                                          esterases stereoselective synthesis and Kobayashi et al., Tetrahedron                                   Letters                                              resolution Vol 25, #24: 2557-2560 (1984)                                       Schneider et al., Agnew. Chem. Int.                                           Ed. Engl. 23 (#1): 64-68 (1984)                                             tyrosinase oxidation of phenols to produce Kazandjian, R. Z. and                                        Klibanov, A. M.                                      quinones J. Am. Chem. Soc. 110: 584-589 (1986)                               proteases, eg subtilisin stereoselective acylation of Riva et al. J.                                    Am. Chem. Soc. 110: 584-                             carbohydrates 589 (1988)                                                     oxidases selective oxidation of hydrocarbons Klibanov, A. M. Acc. Chem.                                 Res. 23:                                              114-120 (1990) and references therein                                       other enzymes not Stereoselective synthesis: Wong, C. H., Chemtracts-Org                                anic                                                requiring co-factors:  Chemistry 3: 91-111 (1990) and                         isomerases, lyases,  references therein                                       aldolases, glycosyl                                                           transferases, glycosidases                                                    other enzymes not Stereoselective synthesis: Wong, C. H., Chemtracts-Org                                anic                                                requiring added co-factors:  Chemistry 3: 91-111 (1990) and                   flavoenzymes, pyridoxal  references therein                                   phosphate enzymes,                                                            metalloenzymes                                                                enzymes requiring co- Stereoselective synthesis: Wong, C. H., Chemtracts                                -Organic                                            factors: kinases (ATP),  Chemistry 3: 91-111 (1990) and                       oxidoreductatses (NAD/P),  references therein                                 methyl transferases                                                           (SAM), CoA-requiring                                                          enzymes, sulfurases                                                           (PAPS)                                                                      __________________________________________________________________________

Schneider et al. (Agnew. Chem. Int. Ed. Engl. 23 (No.1): 64-68 (1984))illustrates how enzymes may be used in organic syntheses. Pig liveresterase was employed in the meso-ester transformation into a chiralmono-ester in an aqueous phosphate buffer.

The advantages of CLEC catalyzed reactions for laboratory use arethreefold. First, CLECs retain high activity in harsh environments (eg.aqueous, organic, near-anhydrous solvents and mixtures of these, and athigh temperatures) that are typical of laboratory chemical synthesisexperiments. Second, CLEC exhibit high operational and storagestability, which is appropriate for intermittent laboratory experiments.Third, their high activity per unit volume will allow shorter reactiontimes and require smaller volumes of enzyme (per unit of activity).Thus, the advantages that CLECs offer over free or immobilized enzymes,provide organic chemists with an alternative, highly selective,synthetic tool.

Enzymes are catalytic proteins that make possible or greatly acceleratealmost every biologically important chemical reaction. Enzymes performoxidations, reductions, additions, eliminations, rearrangements,hydrolyses, dehydrations. They are thus responsible for manufacturingevery biologically significant molecule in plants and animals, fromsub-cellular organelles to bones and organ systems. Because an enzymetypically carries out one chemical step at a time in what may be acomplex, multistep transformation (requiring twenty or more enzymes),chemists have for decades been intrigued by the potential for usingenzymes as synthetic chemical catalysts.

Despite the great potential for using enzymes in synthetic chemistry,their utility outside living systems has been hampered by seeminglyinsurmountable problems:

Isolation and purification of enzymes is difficult

Enzymes are susceptible to degradation or inactivation by air, changesin pH, other enzymes, and organic compounds such as solvents

The cost of enzymes and their chemical cofactors (small moleculesrequired for enzymatic action) is high

Relatively little is known about the interactions between enzymes andorganic molecules which are not their natural substrates

Fortunately, biotechnology is beginning to solve problems associatedwith isolation, purification, and cofactor regeneration, and innovativechemists are compiling data almost daily on adapting known enzymes tonew substrates. However, the instability of enzymes and their aversionto solvents other than water have remained barriers to widespread use ofthese proteins in routine organic synthesis.

A CLEC (cross-linked enzyme crystal) contains more than one proteinmolecule organized in a crystalline lattice, which is either crosslinkedor not crosslinked, or crosslinked by simple bifunctional organicmolecules. A typical crosslinking agent, glutaraldehyde, contains twosites for attaching free amino groups found on proteins. As long as theamino group that attaches to glutaraldehyde is far from the enzyme'sactive site, crosslinking has no appreciable effect on enzymaticactivity. After forming, the CLEC may be freeze-dried, air dried or leftin a liquid state. In any of these storage conditions they may be storedindefinitely as a solid at room temperature. But most importantly, CLECsretain their high activity in real-world chemical reaction conditions,including harsh temperatures and pH, in many types of organic or mixedaqueous/organic solvent, and even in the presence of other enzymes thatdigest proteins.

Since so many industrial applications of enzymes depend on themolecule's stability and activity in sub-optimal conditions, CLECs willgreatly expand the use of industrial and research enzymes, as well asnon-enzyme proteins and a wide range of peptides. CLECs replace manyconventional enzymes in applications where soluble or immobilizedenzymes are already used, as well as in new applications where theycannot be used, e.g., in organic solvents or in mixed solutions of waterand non-polar or polar organics, such as acetone, dioxane, acetonitrile,and tetrahydrofuran. Some potential chemical uses of CLECs in industryand research include:

organic synthesis of important high-value intermediates and specialtychemicals.

Chiral synthesis and resolution for optically pure pharmaceuticals andspecialty chemicals.

Bioprocessing of products produced through fermentation, as well asnatural products harvested from plants, animals, and insects.

But CLEC technology is not just for enzymes. It is a general method forachieving unprecedented stability and activity in almost any protein andin smaller peptides. The most exciting applications of CLEC technologymay arise not from chemical catalysis in the laboratory, but intherapeutics and diagnostics--both enzymatic and non-enzymatic--in thehuman body, tissues, or fluids.

There is considerable interest among pharmaceutical companies inproteins, protein fragments, and synthetic peptides as drugs anddiagnostics. More and more drugs in research and development arepeptides or "peptide-like" synthetic organic compounds. The trouble withpeptides and peptide-like compounds--whether they are natural orsynthetic--is stability. The human body contains thousands ofproteolytic enzymes (proteases) that break down proteins and peptidesinto smaller and smaller units. Because of degradation by proteases,some peptides with potential therapeutic or diagnostic benefit havebiological half-lives that are too short to enable them to work.Pharmaceutical scientists try to circumvent degradation of synthetic orsemi-synthetic peptides by masking them with chemical groups that confersome protection from proteases. Unfortunately, because of strictrequirements for chemical structure and molecular shape in biochemicalinteractions, the use of chemical protecting groups tends to lower adrug's affinity for target molecules or receptors.

Applied to non-enzymatic peptides and proteins, CLEC technology canproduce therapeutic and diagnostic compounds with unprecedented in vivostability and near-theoretical activity. Crosslinking may allow drugresearchers to use native proteins or peptides and may eliminate theneed, in certain instances, for designing structural analogues orimplementing masking strategies to make drugs more resistant todegradation. Some potential medical uses of CLECs (or CLEC technology)include:

Therapeutic antibodies that bind to and inactivate viruses, bacteria, orproteins; antibodies against inflammatory mediators and mediators ofnerve and tissue destruction; antibodies to messenger chemicals inconditions such as Alzheimer's disease, stroke, nervous system trauma,and autoimmune diseases; catalytic antibodies, which react with theirimmunologic targets after binding to them.

Diagnostic antibodies that bind to their targets to allow detection invitro or in vivo.

Therapeutic proteins and enzymes that replace proteins absent due toacute, chronic, or inherited diseases; enzymes that dissolve blood clotsor cholesterol deposits.

"Super-Inhibitors" of enzymes, receptors, or small molecules made fromfragments of naturally-occurring inhibitor molecules or even fromsynthetic peptides.

Radiology: Proteins or peptides connected to radioactive elements formedical diagnostics (imaging) and therapeutics (radiotherapy); combinedradiotherapy/imaging.

Enzyme therapy for cancer nutrient deprivation, pancreaticinsufficiency, and other enzyme or protein therapies which, by the useof an enzyme or a protein results in the elimination of an adversehealth state, and extracorporeal treatment (in which blood, lymph, orother tissues are removed from the patient, treated, and re-introduced).

Oral peptide drug delivery--conventional peptide drugs are quicklybroken down in the gut, mostly by proteases. The improved stability ofCLECs toward proteolysis may make these compounds attractivealternatives to intravenously administered peptide and peptide-likedrugs.

Other important applications of CLECs include biosensors. Biosensorsinclude many types of devices and technologies that detect and quantifybiologically important events. For our purposes, however, biosensors areimmobilized molecules connected to some type of signaltransducer--either optical, electrical, electromagnetic, orchemical--that produce a signal in the presence of an analytebiomolecule. Many biosensors simply detect the presence (or absence) ofan analyte. More sophisticated sensors also quantify the amount of theanalyte. Commercially available antibody-based biosensors are based onantibody-antigen interactions, one of the most specific chemicalinteractions known between two molecules.

Antibody-based biosensors have the same problems as other technologiesbased on purified enzymes: reduced operational and storage stability,vulnerability to contamination, and susceptibility to degradation whenused in living tissues or on bodily fluids. Other technical hurdles toenzyme biosensors include difficulty in miniaturization, integration ofa biomolecule and transducer, contamination and cross-reactivity.

CLEC technology for biosensors and diagnostics is applicable to enzymeantibodies, catalytic antibodies and other reaction proteins. Theseproducts may include:

Agricultural testing for pesticides, parasites, toxins, drug residues,and food composition.

Environmental monitoring and control.

Medical and veterinary diagnostic tests for home, clinics, hospitals,clinical laboratories, and physician offices.

Industrial process monitoring, primarily in biotechnology.

Real-time monitoring and sensing for research and therapy using animals,tissues, or cells.

CLECs offer many advantages over non-crosslinked enzymes. Theseadvantages also hold for non-enzymatic proteins, and perhaps for smallerpeptides as well:

Superior Stability--The intermolecular contacts and crosslinks betweenenzymes in the crystal lattice of a CLEC stabilize the enzyme andprevent denaturation. CLECs remain active and are resistant toproteolysis, extremes of temperature, and organic solvents. CLECs arestable at room temperature indefinitely.

Solid Form--Soluble enzymes require immobilization in most applications.

CLECs, which can be either soluble or insoluble enzyme particles,eliminate the need for an inert support.

Highest Possible Concentration--the activity, per unit volume, of CLECsis significantly higher than that of conventionally immobilized enzymesor concentrated soluble enzymes. Enzyme concentrations within a CLEC areclose to theoretical limits.

Superior Uniformity--As crystals, individual CLEC particles are uniformand monodisperse, in contrast to undefined, and often random attachmentof conventional enzymes. CLECs remain monodisperse on reconstitution,even in organic solvents.

Operational Convenience--CLECs can easily be freeze-dried or air driedand, in that form, can be stored indefinitely at room temperature.

CLECs have special advantages over soluble or conventionally immobilizedenzymes for bioprocessing, including:

Improved yield under harsh conditions or situations requiring highthroughput, enabling process chemists to concentrate on maximizing yieldwith less concern about reaction conditions.

Cleaner, faster reaction--Because they are not soluble, CLEC enzymes canbe easily separated from the product via settling or filtration, therebyeliminating a source of contamination.

Longer catalyst life and faster reaction times.

Environmentally benign--biocatalysts are easier to dispose of than mostsynthetic catalysts.

Mechanical strength--allowing continuous flow rather than batchprocesses.

As biosensors, CLECs have all the advantages of immobilized enzymes andnone of the drawbacks:

High specificity, sensitivity, and accuracy.

Extended operational lifetime and storage stability.

Miniaturization--Since CLECs are insoluble crystals, they do not requirean inert carrier and exhibit the highest protein density possible.

The high specific activity means a CLEC will generate the largestpossible signal from even the smallest substrate (analyte)concentration.

Uniform Signal--Proteins in crystal form are uniformly arranged. Thisuniformity should produce a linear and predictable signal.

Resistance to Contamination--Soluble enzymes are vulnerable toproteolysis as well as contamination in many biosensor environments.Since CLECs are crystalline they are less susceptible to contaminationand may be reused with minimum effort.

CLECs will have a primary impact on companies involved in industrial andresearch enzymes, medical therapeutics and diagnostics, and biosensors.These firms will now be able to direct their research efforts towardproducing biocatalysts, drugs, and other products with less concern forthe stability of their molecules and more concern for function.

Most CLECs retain nearly 100% of the activity of soluble enzymes aftertwo weeks or more in conditions that denature soluble proteins withinhours. Improvements in efficiency and throughput per weight of proteincan therefore easily reach a hundred-fold or more. That may mean, in theshort term, selling less of an enzyme or antibody compared to thesoluble form. However, the improved performance of CLECs may expand thecommercial potential for proteins as much as ten-fold for existingapplications, and thousands of times for as-yet undiscovered uses.

CLEC technology will cause pharmaceutical and specialty chemicalmanufacturers to re-think the catalytic potential of many enzymesprocesses (and non-enzyme proteins and peptides) that may have beenshelved due to the low efficiency of idealized soluble or supportedproteins, the high cost of multi-phase reactions, or difficulties inproduct isolation or biocatalyst removal.

Pharmaceutical companies concerned about the in vivo proteolysis orunacceptable biological half-life of protein or peptide therapeutics cannow focus on pharmacologic activity rather than on masking peptide bondsfrom circulating degrading enzymes.

Of course, some proteins may not have free primary amine groups tocombine with glutaraldehyde. In others, the free amines may be too closeto the active site or may be required to retain tertiary structurenecessary for activity. So although the prototype CLEC usesglutaraldehyde conjugated with primary amines, this approach may not beappropriate to every protein, enzyme, antibody, or peptide. For thesemolecules, other suitable crosslinkers can be employed.

In general, CLECs will greatly expand the uses of industrial proteins,especially enzymes and antibodies. It is difficult to pinpoint, fromthis vantage point, the improvement in efficiency required to make arun-of-the-mill peptide interesting scientifically. Predicting whichmolecules will turn a profit is even more difficult, especially inhighly regulated healthcare fields. However, with hundred-foldimprovements in protein stability easily attainable via crosslinking,CLECs will resurrect thousands of peptide research programs and greatlyexpand the scope of currently successful research efforts.

Computer databases of proteins and peptides might lead to a nearlylimitless supply of leads for crosslinking, resulting in dozens orhundreds of new products. Modifying refractory proteins through residuescontaining sulfhydryl or hydroxyl groups might be another way tocrosslink proteins that are poor candidates for joining through freeamine groups.

Other applications can include, for example, deposition of crystallizedenzymes, proteins, peptides, or amino acids on an inert substrate. Also,coating can be formed over crystallized amino acid chains bycrosslinking any outer portion of the crystals with a suitablecrosslinking agent, such as an aldehyde or an oligomer of the aldehyde.An example of a suitable aldehyde is glutaraldehyde.

In all of these instances described above, but not limited to these, themethod of this invention can be adapted by one of ordinary skill in theart, to convert a process using a conventionally immobilized enzymecatalyst to the use of a CLEC of the appropriate enzyme. CLECs can notonly replace conventional immobilized enzymes, but can also be used incell mediated transformations.

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way.

EXAMPLE 1 Crystallization and Crosslinking of Thermolysin for Synthesisof the Aspartame Precursor, Z-Asp-Phe-OMe

Crystallization

250 mg of thermolysin from Bacillus thermoproteolyticus was purchasedfrom Boehringer-Mannheim GmbH, and dissolved in 4 ml of 45% dimethylsulfoxide (DMSO) and 55% 1.40 M calcium acetate, 0.50 M sodiumcacodylate at pH 6.5. These starting conditions are similar to thosedescribed by Matthews et. al. for the production of diffraction qualitythermolysin crystals (see, eg., Holmes and Matthews, J. Mol. Biol. 160:623-639 (1982)). The protein solution was then concentrated to 1 ml in aCentricon 10 micro-concentrator. A good yield of microcrystals wasobtained by a process of flash crystallization, now disclosed here, inwhich 1 ml of water, or 1.40 M calcium acetate, 0.50 M sodium cacodylateat pH 6.5, was rapidly injected into either of the thermolysin-DMSOsolutions described above. A shower of hexagonal micro-crystals ofapproximately uniform dimensions (approx. 10⁻¹ mm in length) resultsfrom this process.

Crosslinking of Thermolysin Microcrystals

The protocol used in this specific example of the method of thisinvention is an adaptation of that described by Nakanishi et. al.(Biotechnology 3: 459-464 (1985)), in which protocol, thermolysin wasfirst adsorbed onto a carrier bead composed of the ion-exchange resinAmberlite XAD-7, and subsequently immobilized by cross-linking withglutaraldehyde (Quiocho and Richards, Proc. Natl. Acad. Sci. (U.S.A.)52:833-839 (1964)). In this exemplification, the microcrystals ofthermolysin obtained above were centrifuged and pelleted, and thesupernatant was discarded. 5 ml of 17.5% technical grade glutaraldehyde,in 2.5% DMSO, 0.05M calcium acetate, and 0.025M sodium cacodylate at pH6.5, were then added to the microcrystals. The mixture was incubatedwith gentle agitation at 37° C. for 4 hours. The crosslinking reactionwas stopped by repeated washing of the crystals with 10 ml aliquots ofwater to remove the glutaraldehyde solution. The washed cross-linkedthermolysin crystals constitute the thermolysin CLEC used below as acatalyst.

Synthesis of Z-Asp-Phe-OMe in an Aqueous Solution

5 ml of a thermolysin CLEC suspension were added to a continuousstirred-batch reactor incubated at 37° C. After centrifugation anddecantation of the supernatant, an aqueous reaction mixture was added tothe CLECs. This solution was prepared by mixing 80 mg of Z-L-Asp and 80mg of L-Phe-OMe-HCl in 1 ml of water, with acetic acid added to obtain apH of 7.0. Samples were taken for analysis by HPLC. Table 6 below showsthe HPLC peak height of the Z-L-Asp substrate peak after the indicatedtime of reaction, normalized to 1 at time t=0. Since Z-L-Asp is ratelimiting in this reaction, measuring its depletion is equivalent tomeasuring the appearance of product Z-L-Asp-L-Phe-OMe (Nakanishi et. al.Biotechnology 3: 459-464 (1985)). Table 6 also includes the normalizedpeak height of limiting Z-L-Asp substrate remaining, and an estimate ofthe degree of completion of the reaction. It is clear that the reactionproceeded to about 20% completion within the first 30 seconds andplateaued there. These results are consistent with the observations ofNakanishi et al. (Biotechnology 3: 459-464 (1985)) when usingconventionally immobilized thermolysin in an aqueous reaction mixture asabove, and are attributable to the sparing solubility of theZ-L-Asp-L-Phe-OMe product in water.

                  TABLE 6                                                         ______________________________________                                        Reaction      Peak Height                                                                             Percent                                                 Time (sec) (Normalized) Completion                                          ______________________________________                                         0            1.000                                                             30 0.727 27.3%                                                                60 0.857 14.3%                                                                120  0.940  6.0%                                                              180  0.797 20.3%                                                            ______________________________________                                    

Synthesis of Z-Asp-Phe-OMe in a Near-anhydrous Solution

5 ml of a thermolysin CLEC suspension were added to a continuousstirred-batch reactor incubated at 37° C. After centrifugation anddecantation of the supernatant, a near-anhydrous organic reactionmixture was added to the CLECs. This solution was prepared by mixing 80mg of Z-L-Asp and 240 mg of L-Phe-OMe in 1 ml of 99% ethyl acetate and1% water. Samples were taken for analysis by HPLC. Table 7 below showsthe HPLC peak height of the Z-L-Asp substrate peak after the indicatedtime of reaction, normalized to 1 at time t=0. Since Z-L-Asp is ratelimiting in this reaction, measuring its depletion is equivalent tomeasuring the appearance of product Z-L-Asp-L-Phe-OMe (Nakanishi et. al.Biotechnology 3: 459-464 (1985)). Table #7 also includes the normalizedpeak height of limiting Z-L-Asp substrate remaining, and an estimate ofthe degree of completion of the reaction. In this case, the reactionproceeded to about 70% completion within the first 30 seconds andplateaued there. These results are also consistent with the observationsof Nakanishi et al. (Biotechnology 3: 459-464 (1985)) withconventionally immobilized thermolysin in a near-anhydrous reactionmixture, and are attributable to product inhibition of the enzyme.

                  TABLE 7                                                         ______________________________________                                        Reaction      Peak Height                                                                             Percent                                                 Time (sec) (Normalized) Completion                                          ______________________________________                                         0            1.000                                                             30 0.323 67.7%                                                                60 0.314 68.6%                                                                120  0.305 69.5%                                                              180  0.272 72.8%                                                            ______________________________________                                    

EXAMPLE 2 Crystallization, Cross-Linking and Lyophilization ofThermolysin and Assessment of Characteristics of Resulting Product

Crystallization of Thermolysin

Thermolysin (Diawa Kasei K.K., Japan) was dissolved in 10 mM calciumacetate (Sigma), pH 10.0, to a concentration of 10% (w/v). The pH of thesolution was maintained at 10.0 by titration with 2 M NaOH. Followingcomplete solubilization, the protein solution was titrated to pH 8.0with 2 M HCl. Solid calcium acetate was added to 1.2 M. Dimethylsulfoxide (Sigma) was then added to 30%. The protein was concentrated to100 mg/ml by ultrafiltration in an Amicon stir cell (10,000 MWCOmembrane). Concentrated enzyme was aliquoted and stored at -70° C.Thermolysin was crystallized by the addition of 9 volumes demineralizedwater to 1 volume concentrated (100 mg/ml) protein solution. Thesolution was briefly vortexed and allowed to stand overnight at roomtemperature. Crystals were washed with 10 volumes of 10 Mm calciumacetate pH 7.0 and recovered by low speed centrifugation (10 min at1500×G, Beckman GPR centrifuge).

The rapid addition of water to a concentrated (100 mg/ml) solution ofthermolysin induces the formation of crystals which become visible underlow-power magnification within ten minutes. Crystal size is reproduciblydependent on the final protein concentration. Three volumes of water toone volume of thermolysin concentrate (100 mg/ml) will produce 0.5 mmlong, X-ray diffraction quality hexagonal rods that correspond to thecrystals described earlier by Colman et al. (Colman, P. M., Jansonius,J. N. and Matthews, B. W., J. Mol. Biol. 70: 701-724 (1972)), asconfirmed by us by diffraction analysis. Adding ten volumes of water toone of protein concentrate reduces the length of the resulting crystalsto 0.05 mm. These micro-crystals are preferred in CLEC applications,since they tend to minimize diffusion problems related to crystal size(see eg., Quiocho, F. A. and Richards, F. M. Biochemistry 5: 4062-4076(1967)). Within a given batch of protein, crystal size was consistentlyuniform. (Crystals 0.05-0.10 mm in length were used in this study tofacilitate accurate pipetting of crystalline suspensions.) Densitometerscans of SDS-PAGE showed a six-fold purification of the enzyme oncrystallization, significantly increasing the specific activity of theCLECs. Crystallization resulted in a 20% decrease in the total activityof the CLEC protein compared to soluble thermolysin, when assayed byspectrophotometric cleavage of the dipeptide substratefurylacryloyl-glycyl-L-leucine-amide (FAGLA), as described below.

Crosslinking of Thermolysin Crystals

Thermolysin crystals were crosslinked for 3 hours at room temperature ina solution of 12.5% glutaraldehyde (Sigma), 5% DMSO and 50 mM Tris pH6.5. The crosslinked crystals were washed 3 times in demineralized waterand recovered by low speed centrifugation, as described in respect tocrystallization of thermolysin. Chemical cross-linking of enzymecrystals stabilizes the crystal lattice and the constituent enzymemolecules in the crystal sufficiently so as to permit the practical useof CLECs in environments that are otherwise incompatible with enzymefunction. There was no measurable difference in enzymatic activitybetween the crosslinked and un-crosslinked crystals when assayed(spectrophotometrically) by monitoring cleavage of the dipeptidesubstrate FAGLA (described below). Moreover, cross-linking stabilizesCLECs to the point that they can be lyophilized, with retention of fullenzymatic activity upon reconstitution in aqueous, organic, and mixedaqueous-organic solvents as shown in FIG. 1 and Table 8. Althoughcrystallization resulted in a 30% decrease in the specific activity ofthe CLEC protein compared to soluble thermolysin, crosslinking andlyophilization of the CLECs did not further diminish specific activity.

                  TABLE 8                                                         ______________________________________                                        Thermolysin Activity                                                                           Absorbance 345 nm                                            Time (min)       CLEC    Soluble Enzyme                                       ______________________________________                                        1     0.0            0.314   0.315                                              2 1.0 0.272 0.271                                                             3 3.0 0.235 0.218                                                             4 5.0 0.204 0.198                                                             5 10.0 0.184 0.185                                                            6 15.0 0.183 0.184                                                          ______________________________________                                    

Enzymatic Activity of Soluble and CLEC Thermolysin

The catalytic activity of soluble and CLEC thermolysin was assayed(Feder, J. and Schuck, J. M., Biochemistry 9: 2784-2791 (1970)) byhydrolysis of the blocked dipeptide substratefurylacryloyl-glycyl-L-leucine-amide (FAGLA) (Schweizerhall). Cleavageof the amide bond was measured spectrophotometrically by a decrease inabsorbance at 345 nm. Initial enzyme concentration was 10⁻⁷ M byBradford protein determination and densitometer scanning (Pharmacia LKBUltroScan XL) of Coomassie stained SDS-PAGE gels. CLEC enzyme is definedas reconstituted lyophilized crosslinked thermolysin crystals. Solubleenzyme is defined as thermolysin concentrated to 100 mg/ml. Enzyme wasadded to a 5 ml reaction volume containing substrate. Aliquots of thereaction mix were removed at the indicated times, and absorbance at 345nm was measured. CLEC thermolysin was separated from the reaction mix bybrief centrifugation (Beckman, microcentrifuge E) before readingabsorbance. Absorbance was fitted to a pseudo first order rate equationand kcat/Km was calculated by dividing the fitted value by enzymeconcentration (Multifit 2.0 Curve Fitting for the Apple MacintoshComputer, Day Computing P.O. Box 327, Milton, Cambridge CB4 6WL, U.K.(1990)).

pH Dependence and Stability

The pH optimum and stability of the soluble enzyme were compared to thatof thermolysin CLECs by cleavage of the dipeptide substrate FAGLA.Results are shown in FIG. 2 and Table 9. Both soluble and crystallineenzyme forms demonstrate maximum activity at pH 7. CLECs and solublethermolysin also demonstrated similar activity in the acidic range andthe bell shaped pH profile generated by the soluble enzyme was in goodagreement with published data (Feder, J. and Schuck, J. M., Biochemistry9: 2784-2791 (1970)). In the alkaline pH range, however, the crystallineenzyme maintains maximum activity, to pH 10, while the soluble enzymehas 75% activity at pH 8.5, and only 25% activity at pH 9. At pH 9.5,the soluble enzyme is completely inactive.

                  TABLE 9                                                         ______________________________________                                        Thermolysin pH Curve                                                                          % Maximum Activity                                            pH              CLEC    Soluble Enzyme                                        ______________________________________                                        1       5.0         10.250  5.170                                               2 5.5 9.750 6.070                                                             3 6.0 52.500 39.100                                                           4 6.5 85.000 74.610                                                           5 7.0 97.500 100.000                                                          6 7.5 100.000 98.650                                                          7 8.0 97.500 82.920                                                           8 8.5 95.000 71.910                                                           9 9.0 96.250 24.720                                                           10   9.5 95.000 0.000                                                         11  10.0 90.000 0.000                                                       ______________________________________                                    

Stability at Elevated Temperature

One can achieve higher reaction rates and lower diffusion times forsubstrates and products by operating a given chemical process at highertemperature, where one is usually limited by the temperature stabilityof substrates and products. In enzyme-based catalysis, however, it isoften the loss of enzymatic activity that sets the practical limit onthe temperature that a process can be run. The additional stabilizationachieved in CLECs allows for enzymatic activity at much highertemperatures than is possible for soluble enzyme.

The enhanced stability at lower temperatures simplifies the routine longterm storage of the CLEC catalysts. For example, it was necessary tostore concentrated (>50 mg/ml) solutions of soluble thermolysin at -80°C. to retain maximum specific activity. At room temperature, activitywas usually lost within one day. In contrast, rehydrated thermolysinCLECs could be routinely stored-for months at room temperature with noapparent loss of activity. Unreconstituted lyophilized CLECs ofthermolysin appear to be viable indefinitely.

Thermal stability and resistance to autolysis were demonstrated inthermolysin CLECs following incubation at 65° C. for five consecutivedays (FIG. 3 and Table 10). Thermolysin CLECs retained maximum activityafter five days incubation at elevated temperature. In contrast, thesoluble thermolysin lost 50% of its initial activity after only twohours incubation and demonstrated negligible activity after 24 hoursincubation at 65° C.

                  TABLE 10                                                        ______________________________________                                        Thermolysin Thermal Stability at 65° C.                                       Time         % Maximum Activity                                        (days)          CLEC    Soluble Enzyme                                        ______________________________________                                        1      0.000        100.000 100.000                                             2 0.041  70.000                                                               3 0.083 96.000 50.000                                                         4 0.164  32.000                                                               5 0.246  17.000                                                               6 0.410 97.0 10.000                                                           7 1.000 101.0 2.000                                                           8 2.000 97.0                                                                  9 3.000 94.0                                                                  10   4.000 96.0                                                               11  5.000 92.0                                                              ______________________________________                                    

The activity of soluble and CLEC thermolysin was measured followingincubation at 65° C. Soluble thermolysin was incubated in 10 mM calciumacetate, 50 mM Tris pH 7.0 in a 65° C. water bath. The reaction volumewas 500 μl. Final protein concentration was 10 mg/ml. Aliquots wereremoved at times 0, 1, 2, 4, 6, 10, and 18 hours. The samples wereassayed by SDS-PAGE and FAGLA cleavage at room temperature as describedabove. For the thermolysin CLECs, a 250 μl crystal suspension in 10 mMcalcium acetate and 50 mM Tris was also incubated in a 65° C. waterbath. Activity was assayed at times 0, 1, 6, 24, 48, 72, 96, and 120hours by FAGLA cleavage.

Resistance to Exogenous Proteolysis

Assessment of the resistance of the thermolysin CLEC to the action of anexogenous protease was also carried out. SDS-PAGE (Sodium dodecylsulfate poly acrylamide gel electrophoresis) analysis suggests thatcommercial enzymes can contain a substantial percentage of contaminants,some of which might have proteolytic activity against the principalsoluble enzyme species. Given the packing of enzyme molecules in acrystal lattice one might assume that the interior enzyme molecules in aCLEC would be protected from proteolysis. To test this possibility,thermolysin CLECs and a soluble enzyme preparation were incubated in thepresence of the streptococcal protease, Pronase®, a nonspecific proteasecapable of digesting most proteins to free amino acids (Calbiochem 1990Catalog; LaJolla, Calif.).

Soluble and CLEC thermolysin were incubated in 50 mM Tris, pH 7.5, at40° C. in the presence of the protease Pronase® (Calbiochem). ThePronase® to thermolysin ratio was 1/40. To inhibit thermolysin autolysisand prevent the proteolytic destruction of pronase by the thermolysin,EDTA was added to the soluble enzyme reaction to a final concentrationof 100 mM (EDTA inhibits thermolysin activity but not Pronase®). At thetimes indicated aliquots were removed from the reaction mix and activitywas assayed spectrophotometrically by cleavage of the dipeptidesubstrates FAGLA. To offset thermolysin inhibition due to the presenceof EDTA, the spectrophotometric assay of soluble enzyme activity wasperformed in 0.5 M calcium acetate buffer pH 7.0 and enzymeconcentration was increased two fold. Crosslinked crystalline enzyme wasassayed as described above.

As can be seen in FIG. 4 and Table 11, the soluble thermolysin wasrapidly degraded and lost all activity after 90 minutes incubation. Incontrast, the activity of the thermolysin CLEC was unaffected by fourdays incubation in the presence of protease. This near imperviousness toproteolysis is of particular interest in diagnostic biosensorapplications where a suitable CLEC might be called upon to act in thepresence of an unknown cocktail of naturally occurring proteolyticenzymes.

                  TABLE 11                                                        ______________________________________                                        Protease Resistance                                                                   Time         % Maximum Activity                                                                            Time                                     (days)      CLEC       Soluble Enzyme                                                                            (min)                                      ______________________________________                                        1       0.000   100.0      100.0      0.000                                     2 0.003  25.0  5.000                                                          3 0.010  17.5 15.000                                                          4 0.021  9.5 30.000                                                           5 0.042 98.0 3.0 60.000                                                       6 0.063  1.0 90.000                                                           7 0.084 101.0 0.0                                                             8 1.000 97.0                                                                  9 2.000 99.0                                                                  10  3.000 98.0                                                                11  4.000 96.0                                                              ______________________________________                                    

Stability in the Presence of Organic Solvent

In order for enzymes to gain ideal acceptance as viable industrialcatalysts, they must be able to function without excessive interventionin the practical environment of manufacturing processes. In particular,this would include the use of aqueous, polar and non-polar organicsolvents, and mixtures of these. In commercial applications,aqueous-organic solvent mixtures allow manipulation of product formationby taking advantage of relative solubilities of products and substrates.

Soluble thermolysin and thermolysin CLECs exhibited markedly differentstability in the presence of organic solvents. (Table 12). Solubleenzyme concentrations which could be incubated in organic solvent werelimited to a maximum of 10 mg/ml. Concentrations greater than this valueresulted in the instantaneous precipitation of thermolysin upon additionof organic solvent. In contrast, thermolysin CLEC concentrations werelimited only by the volume occupied by the crystals. Soluble thermolysinretained the greatest activity (75%) following incubation in acetone,and the least (36%) in tetrahydrofuran. Following a one hour incubationin the presence of acetonitrile or dioxane the soluble enzyme lostapproximately 50% of its initial activity. The CLEC thermolysin retainedgreater than 95% maximum activity following incubation with all organicsassayed.

                  TABLE 12                                                        ______________________________________                                                     % Maximum Activity                                                            Soluble Enzyme                                                                          CLEC                                                   ______________________________________                                        Acetonitrile   42          102                                                  Dioxane 66 97                                                                 Acetone 75 99                                                                 THF* 36 96                                                                  ______________________________________                                         *Tetrahydro Furan                                                        

Stability in Organic Solvents

Thermolysin CLECs or soluble thermolysin preparations were incubated in50% (v/v) solutions of the indicated organic solvents. A 100 μl slurryof thermolysin CLECs (10 mg/ml) in 10 mM Tris pH 7 was placed in a 1/2dram glass vial. An equal volume of the indicated organic solvent wasadded and the mixture was briefly vortexed. Twenty μl of solublethermolysin (100 mg/ml) was diluted in 80 μl of 0.015M Tris buffer pH7.0 in a 1/2 dram glass vial. A 100 μl volume of organic solvent wasthen added to the protein solution and briefly vortexed. CLEC andsoluble enzyme were incubated in the presence of organic solvent for onehour at 40° C. Following incubation, enzyme activity was assayed bycleavage of the dipeptide substrate FAGLA as described.

Low water concentration is thought to disfavor unfolding to intermediatestates on the path to enzyme denaturation. In CLECs, this restriction ofconformational mobility is provided by the inter-molecular contacts andcross-links between the constituent enzyme molecules making up thecrystal lattice, rather than by the near-absence of water in the medium.As a result, intermediate water-organic solvent concentrations arereadily tolerated by enzymes when formulated as CLECs, somethingpreviously unobserved with enzymes (see Table 12). This discovery opensup whole new areas of synthetic chemistry to exploitation using enzymecatalysis.

Even in near-anhydrous organic solvents, however, the routine use ofenzymes has been hampered by their tendency to form ill-definedsuspensions that are subject to clumping and other aggregation problems.This property makes these preparations inherently unattractive for largescale industrial processes. In contrast, CLECs and the constituentenzymes within the crystal lattice, remain mono-disperse in all thesesolvents.

Comparison With Other Immobilization Methods

A number of useful reviews of enzyme immobilization methods haveappeared in the literature (Maugh, T. H., Science, 223: 474-476 (1984));Tramper, J., Trends in Biotechnology 3: 45-50 (1985)). In these, theenzyme always represents a small fraction of the total volume of theimmobilized particle, the bulk of it being inert carrier material. Thecarrier increases the mean free path between the solvent exterior of theimmobilized enzyme particle and the enzyme active sites, exacerbatingdiffusion problems (Quiocho, F. A. and Richards, F. M., Biochemistry 5:4062-4076 (1967)).

In a CLEC, the crosslinked crystal matrix provides its own support,eliminating the need for a carrier. As a result, the concentration ofenzyme in a CLEC is close to the theoretical packing limit that can beachieved for molecules of a given size, greatly exceeding densitiesachievable even in concentrated solutions. The entire CLEC consists ofactive enzyme, and thus, the diffusion-related reduction of enzymereaction rates usually observed with conventionally immobilized enzymesrelative to enzymes in solution are minimized (See FIG. 1), since themean free path for substrate and product between active enzyme and freesolvent will be greatly shortened for CLECs (compared to a conventionalimmobilized enzyme carrier particles). Importantly, the constituentenzyme in CLECs is intrinsically mono-disperse, and can be recovered bysimple manipulations of the CLEC particles, such as filtration,centrifugation or decantation of solvent.

EXAMPLE 3 Soluble and CLEC Thermolysin-Catalysed Synthesis of theAspartame Precursor Z-Asp-Phe-OMe

Soluble and CLEC thermolysin catalysed synthesis of the aspartameprecursor was performed in a repeated batch experiment. Three differentexperimental parameters were assessed: 1. thermolysin CLEC versussoluble thermolysin half-life, 2. thermolysin CLEC versus solublethermolysin specific activity (equivalent protein concentration) and 3.thermolysin CLEC versus soluble thermolysin total activity (equivalentprotein dry weight).

The reagents were prepared as follows:

Solvent--a buffer (50 mM MES-NaOH, 5 mM CaCl₂(2-[N-morpholino]ethane-sulfonic acid)(Sigma) saturated ethyl acetate,pH 6.0) saturated solution of ethyl acetate (Nakanishi, K. et al.,Biotechnology 3:459-464 (1985)). The MES buffer was prepared bydissolving 9.76 g MES and 0.102 g CaCl₂ in 90 ml deionized water. The pHwas adjusted to 6.0 with 5 N NaOH. The volume was adjusted to 100 ml. Toprepare buffer saturated ethyl acetate, 10 ml MES buffer was combinedwith 90 ml ethyl acetate in separatory funnel, following agitation theorganic phase was collected.

Substrates--240 mM L-Phe-O-Me, 80 mM CBZ-L-Aspartic Acid. L-Phe-O-Me wasprepared by chloroform extraction of L-Phe-O-Me HCl. Equimolar amountsof L-Phe-O-Me HCl and Na₂ CO₃ were dissolved in deionized water andagitated with an appropriate amount of chloroform to extract theL-Phe-O-Me. The chloroform was dehydrated with an appropriate amount ofMgSO₄ and evaporated at 40° C. L-PheOMe was stored as a 2.4 M solutionin ethyl acetate at -20° C.

Thermolysin CLECS--0.216 mM thermolysin CLECs (7.5 mg/ml enzyme,approximately 15 mg/ml dry weight).

Procedure for 3 ml Batch Reaction

0.214 g of N-CBZ-L-Asp was dissolved in 9 ml buffer saturated ethylacetate. 1 ml 2.4 M Phe-O-Me stock as described above was added to theCBZ-aspartic acid. Lyophilized CLECs or lyophilized soluble thermolysinwas added to 3 ml of the reaction mix containing buffered solvent andsubstrates. The reaction was incubated at 55° C. with agitation, pH wasmaintained at 6.0. The CLECs remained insoluble and were removed fromthe reaction by filtration and low speed centrifugation. The conversionof substrates to product was monitored by removing 0.1 ml of reactionmix at 1 hr intervals for TLC or HPLC. The reaction volumes were scaledto 19 ml for the 24 hour continuous batch reactions.

Assay of Aspartame Precursor Product

The progress of the reaction was followed by thin layer chromatography(TLC) (Lindeberg, G, J. Chem. Education 64:1062-1064 (1987)) (mobilrphsdr; 1:1:3 water:acetic acid:N-butanol). Solid phase; silica gel.Visualize by UV at 245 nm and ninhydrin, and by high performance liquidchromatography (HPLC) (Nakanishi, K. et al. Biotechnology 3:459-464(1985); Oyama, K. et al., Meth. in Enzymology 136:503-516 (1984);Ooshima, H. et al., Biotechnology Letters 7:789-792 (1985)). Thereaction was monitored at 214 and 280 n. One hundred per cent conversionto product is defined as one hundred per cent conversion of CBZ-asparticacid to aspartame precursor. Continuous batch synthesis of the aspartameprecursor (half-life)

Soluble and CLEC thermolysin catalysed synthesis of the aspartameprecursor was performed continuously for 10 days in a repeated 24 hourbatch experiment under the conditions described above. CLEC thermolysinconcentration was 15 mg/ml dry weight. Soluble thermolysin (Diawa 10%protein) concentration was 37.5 mg/ml dry weight. Product was recoveredand fresh substrate was added to the reaction every 24 hr. Enzyme wasrecovered from the reaction mix by centrifugation and filtration. The %product was assayed by TLC and HPLC as described above (FIG. 5).

Batch Synthesis Data (Total Activity)

    ______________________________________                                                       % Product                                                      Time (hours)     CLECs   Soluble                                              ______________________________________                                        0                 0      0                                                      1 27 0                                                                        2 63 3                                                                        3 95 5                                                                        4 100  7                                                                    ______________________________________                                    

EXAMPLE 4 Crystallization, Cross-Linking and Lyophilization of Elastaseand Assessment of Characteristics of the Resulting Product

Crystallization of Elastase

Lyophilized porcine pancreatic elastase (Serva) was dissolved in 0.1 Msodium acetate pH 5.0 to a concentration of 5 mg/ml (w/v) at roomtemperature. Rod shaped elastase crystals were visible within one minuteof the complete solvation of the protein. The crystallization solutionwas transferred to 4° C. and crystallization was completed overnight.Crystals were recovered by centrifugation as previously described.

Cross-Linking of Elastase Crystals

A 200 μl volume of elastase crystals was added to a 1.3 ml solution of5.77% glutaraldehyde and 1.5 M sodium acetate pH 5.0. The crystals werecrosslinked for one hour with mild agitation (stir plate). Followingcross-linking the crystals were washed with three 15 ml volumes of 0.2 MTris pH 8.0. The elastase CLEC was lyophilized as described in Example2.

Enzymatic Activity of Soluble and CLEC Elastase

The catalytic activity of soluble and CLEC elastase was assayedspectrophotometrically by measuring hydrolysis of the substratesuccinyl--(Ala)₃ p-nitroanilide (Bachem) [Bieth, et al. Biochem. Med11:350-357 (1974)] (Table 13, FIG. 6). Cleavage was monitored byincreasing absorbance at 410 nm. Initial substrate concentration was2×10⁻⁴. Enzyme concentration was 2.8×10⁻⁷ M. CLEC or soluble enzyme wasadded to a 5 ml reaction volume containing substrate in 0.2 M Tris pH8.0. As described previously, CLEC enzyme was removed from the reactionmix prior to measuring absorbance.

                  TABLE 13                                                        ______________________________________                                        Elastase Activity                                                                              Absorbance 400 nm                                            Time (min)       CLEC    Soluble Enzyme                                       ______________________________________                                        1      0.0           0.000   0.000                                              2 0.5 0.103 0.205                                                             3 1.0 0.195 0.390                                                             4 2.0 0.366 0.672                                                             5 3.0 0.523 0.923                                                             6 4.0 0.657 1.098                                                             7 5.0 0.780 1.227                                                             8 6.0 0.888 1.326                                                             9 7.0 0.974 1.393                                                             10   10.0 1.170 1.512                                                         11  15.0 1.365 1.586                                                        ______________________________________                                    

Resistance to Exogenous Proteolysis

Assessment of the resistance of the elastase CLEC to the action ofprotease was also performed under identical conditions as described forthermolysin (Example 2). Activity of the soluble and CLEC enzyme,following incubation with protease, was assayed by hydrolysis of thenitroanilide substrate as described above (Table 14 and FIG. 7).

                  TABLE 14                                                        ______________________________________                                        Elastase Resistance to Proteolysis                                                           % Maximum Activity                                             Time           CLEC    Soluble Enzyme                                         ______________________________________                                        1      0.0         100.0   100.0                                                2 10.0  53.0                                                                  3 20.0  32.0                                                                  4 30.0 101.0 18.0                                                             5 45.0  11.0                                                                  6 60.0 102.0 8.0                                                              7 120.0 101.0 3.0                                                             8 180.0 103.0 2.0                                                           ______________________________________                                    

EXAMPLE 5 Crystallization, Cross-Linking and Lyophilization of Esteraseand Assessment of Characteristics of the Resulting Product

Crystallization of Esterase

As disclosed here, 30 mg/ml ammonium sulfate suspension of pig liveresterase (Fluka) was dissolved in 0.25 M calcium acetate pH 5.6 at roomtemperature. Esterase crystals were visible within several minutesfollowing addition of the calcium acetate solution. The crystallizationsolution was allowed to stand at room temperature and crystallizationwas completed overnight. Crystals were recovered by centrifugation aspreviously described in Example 2.

Cross-Linking of Esterase Crystals

As disclosed here, a 300 μl volume of esterase crystals were added to a5 ml solution of 12.5% glutaraldehyde and 0.5 M sodium acetate pH 5.5.The crystals were crosslinked for one hour with mild agitation (stirplate). Following cross-linking the crystals were washed with three 15ml volumes of 0.5 M calcium acetate pH 6.3. The esterase CLEC waslyophilized as previously described in Example 2.

Enzymatic Activity of Soluble and CLEC Esterase

The catalytic activity of soluble and CLEC esterase was assayedspectrophotometrically by monitoring hydrolysis of the substratep-nitrophenyl acetate (Fluka) (Table 15 and FIG. 8). Cleavage wasmonitored by increasing absorbance at 400 nm. Initial substrateconcentration was 0.001%. Enzyme concentration was 1×10⁻⁸ M. CLEC orsoluble enzyme was added to a 5 ml reaction volume containing substratein 0.25 M calcium acetate pH 6.3. As described previously in Example 2,CLEC enzyme was removed from the reaction mix by centrifugation prior tomeasuring absorbance.

                  TABLE 15                                                        ______________________________________                                        Esterase Activity                                                                              Absorbance 400 nm                                            Time (min)       CLEC    Soluble Enzyme                                       ______________________________________                                        1     0.0            0.000   0.000                                              2 0.5 0.770 0.252                                                             3 1.0 0.128 0.297                                                             4 2.0 0.208 0.337                                                             5 3.0 0.260 0.346                                                             6 5.0 0.324 0.353                                                             7 7.0 0.353 0.359                                                             8 10.0 0.369 0.368                                                          ______________________________________                                    

Resistance to Exogenous Proteolysis

Assessment of the resistance of the esterase CLEC to the action ofprotease was also performed under identical conditions as described forthermolysin (Example 2). Activity of the soluble and CLEC enzyme,following incubation with protease, was assayed by hydrolysis of thesubstrate p-nitrophenyl acetate as described above (Table 16 and FIG.9).

                  TABLE 16                                                        ______________________________________                                        Esterase Resistance to Proteolysis                                                             % Maximum Activity                                           Time (min)       CLEC    Soluble Enzymes                                      ______________________________________                                        1     0.0            100.0   100.0                                              2 10.0  68.0                                                                  3 20.0  47.0                                                                  4 30.0 99.0 25.0                                                              5 45.0  20.0                                                                  6 60.0 97.0 16.0                                                              7 120.0 94.0 10.0                                                             8 180.0 91.0 6.0                                                            ______________________________________                                    

EXAMPLE 6 Crystallization, Cross-Linking and Lyophilization ofGeotrichum Candidum Lipase and Assessment of Characteristics of theResulting Product

Crystallization of Lipase

As disclosed here, the enzyme lipase (Geotrichum (G.) candidum) wascrystallized by vapor diffusion from an aqueous solution of 20 mg/mlprotein in 50 mM Tris pH 7 containing 8% ammonium sulfate. Bipyrimidalcrystals were visible after 20 to 30 days incubation at roomtemperature. Crystals were recovered by centrifugation, as previouslydescribed in Example 2.

Cross-Linking of Lipase Crystals

As disclosed here, lipase crystals were added to a solution of 12.5%glutaraldehyde and 50 mM Tris pH 5.6. The crystals were crosslinked forone hour. Following cross-linking the crystals were washed with three 15ml volumes of 50 mM Tris pH 7.0. The lipase CLEC was lyophilized, aspreviously described in Example 2.

Enzymatic Activity of Soluble and CLEC Lipase

The catalytic activity of soluble and CLEC lipase was assayedspectrophotometrically by monitoring hydrolysis of the substratep-nitrophenyl acetate (Table 17 and FIG. 10). Cleavage was monitored byincreasing absorbance at 400 nm. Initial substrate concentration was0.005%. Enzyme concentration was 1.5×10⁻⁸ M. CLEC or soluble enzyme wasadded to a 5 ml reaction volume containing substrate in 0.2 M Tris pH7.0 at room temperature. As described previously in Example 2, CLECenzyme was removed from the reaction mix by centrifugation prior tomeasuring absorbance.

                  TABLE 17                                                        ______________________________________                                        Lipase Activity                                                                                Absorbance 400 nm                                            Time (min)       CLEC    Soluble Enzyme                                       ______________________________________                                        1     0.0            0.000   0.000                                              2 1.0 0.013 0.021                                                             3 5.0 0.094 0.116                                                             4 10.0 0.164 0.186                                                            5 15.0 0.248 0.258                                                            6 30.0 0.346 0.357                                                            7 45.0 0.407 0.420                                                            8 60.0 0.461 0.459                                                            9 90.0 0.497 0.502                                                          ______________________________________                                    

EXAMPLE 7 Crystallization, Cross-Linking and Lyophilization of Lysozymeand Assessment of Characteristics of the Resulting Product

Crystallization of Lysozyme

Following the method of Blake, C. C. F. et al., Nature 196:1173 (1962)200 mg of lyophilized hen egg white lysozyme (Boehringer Mannheim) wasdissolved in 2.5 ml of 0.04 M sodium acetate buffer pH 4.7 at roomtemperature. Following solvation of the protein, 2.5 ml of 10% sodiumchloride were added to the lysozyme solution dropwise with stirring. Thecrystallization solution was allowed to stand overnight at roomtemperature, and crystallization was completed in forty-eight hours.Crystals were recovered by centrifugation, as previously described inExample 2.

Cross-Linking of Lysozyme Crystals

As described here, a 500 μl volume of lysozyme crystals was added to 10ml of 24% glutaraldehyde and 50 mM Tris pH 5.6 containing 20% sodiumchloride. The crystals were crosslinked for 20 minutes with mildagitation (stir plate). Following cross-linking the crystals were washedwith three 50 ml volumes of 20 mM calcium acetate and 50 mM potassiumchloride pH 5.3. The lysozyme CLEC was lyophilized, as previouslydescribed in Example 2.

Enzymatic Activity of Soluble and CLEC Lysozyme

The catalytic activity of soluble and CLEC lysozyme was assayed bymeasuring the rate of hydrolysis of the substrate 4-methylumbelliferylN-acetyl-chitrioside (Fluka) (Yang, Y. and Hamaguchi, K. J. Biochem.8:1003-1014 (1980) (Table 18 and FIG. 11). The release of4-methylumbelliferone was followed fluorimetrically (Perkin Elmer ModelLS-50). Initial substrate concentration was 1.42×10⁻³. Enzymeconcentration was 3×10⁻⁷. CLEC or soluble enzyme was added to a 2 mlreaction volume containing substrate in 20 mM calcium acetate and 50 mMpotassium chloride pH 5.3 at 420c. The amount of 4-methylumelliferonewas determined fluorimetrically by measuring fluorescence intensities at450 nm with excitation at 360 nm. Slit width for both excitation andemission was 10 mm. As described previously in Example 2, CLEC enzymewas removed from the reaction mix by centrifugation prior to measuringfluorescence.

                  TABLE 18                                                        ______________________________________                                        Lysozyme Activity                                                                              Fluorescence                                                 Time (min)       CLEC    Soluble Enzyme                                       ______________________________________                                        1     0.000          0.000   0.000                                              2 10.000 4.400 18.900                                                         3 30.000 10.500 29.400                                                        4 60.000 27.500 44.800                                                        5 90.000 33.800 51.700                                                        6 120.000 45.900 59.800                                                     ______________________________________                                    

EXAMPLE 8 Crystallization, Cross-Linking and Lyophilization ofAsparaginase and Assessment of Characteristics of the Resulting Product

Crystallization of Asparaginase

As a modification of the procedure described by Grabner et al. [U.S.Pat. No. 3,664,926 (1972)] 25 mg of lyophilized asparaginase(Worthington) were dissolved in 500 μl of 50 mM sodium phosphate bufferpH 7.2. The solution was cooled to 4° C. and the pH adjusted to 5.0 with1 M acetic acid. Cold (-20° C.) ethanol was then added dropwise to theasparaginase solution to a final concentration of 33%. The solution wasincubated at 4° C. Crystallization was completed in forty-eight hours.Crystals were recovered by centrifugation as previously described.

Cross-Linking of Asparaginase Crystals

As disclosed here, asparaginase crystals were crosslinked in a solutionof 7.5% glutaraldehyde in 50 mM sodium phosphate buffer pH 5.6.Following crosslinking the crystals were washed with five 15 ml volumesof 50 mM tris pH 7.0. The asparaginase CLECs were lyophilized, aspreviously described in Example 2.

Enzymatic Activity of Soluble and CLEC Asparaginase

The catalytic activity of soluble and CLEC asparaginase was assayedspectrophotometrically by measuring evolution of ammonium ion in thecoupled enzymatic reaction described below (all reagents were purchasedfrom Boehringer Mannheim) (Table 19 and FIG. 12). ##STR1## Oxidation ofNADH was measured by decreasing absorbance at 340 nm. Initial NADHconcentration was 1.4 mg/ml. Asparagine concentration was 10⁻³ M. Alphaketoglutarate concentration was 10⁻⁴ M. Glutamate dehydrogenaseconcentration was 10⁻⁷ M. Asparaginase concentration was 2.3×10⁻⁸ M. Asdescribed previously in Example 2, CLEC enzyme was removed from thereaction mix by centrifugation prior to measuring absorbance.

                  TABLE 19                                                        ______________________________________                                        Asparaginase Activity                                                                          Absorbance 340 nm                                            Time (min)       CLEC    Soluble Enzyme                                       ______________________________________                                        1     0.0            1.000   1.000                                              2 1.0 0.867 0.825                                                             3 3.0 0.739 0.684                                                             4 5.0 0.603 0.538                                                             5 10.0 0.502 0.406                                                            6 15.0 0.449 0.338                                                            7 30.0 0.328 0.199                                                            8 45.0 0.211 0.187                                                          ______________________________________                                    

EXAMPLE 9 Crystallization, Crosslinking and Lyophilization of Urease andAssessment of Characteristics of the Resulting Product

Crystallization of Urease

Fifteen thousand units (approximately 180 mg) of lyophilized Jack Beanurease (Boehringer Mannheim) were dissolved in 3 ml of 150 mM sodiumphosphate buffer pH 6.8. Acetone was added to the 3 ml urea solution byovernight vapor diffusion against 80 ml of 50% acetone/50% water. Thesolution was stirred gently during the addition of acetone.Crystallization was completed in sixteen hours. Crystals were recoveredby centrifugation as previously described. Urease crystals were washedtwo times with 40% acetone in 50 mM sodium phosphate buffer pH 6.8.Crystals were recovered by centrifugation following washing.Crystallization resulted in a 40% decrease in the total activity of theCLEC urease compared to soluble urease, when assayedspectrophotometrically, as described below.

Cross-Linking of Urease Crystals

As disclosed here, urease crystals were crosslinked in a solution of 2%glutaraldehyde, 30% acetone in 50 mM sodium phosphate buffer pH 6.Following cross-linking the crystals were washed with four, one litervolumes of 50 M sodium phosphate buffer pH 6.8. Washed crystals weresuspended in deionized water and lyophilized as previously described inExample 2.

                  TABLE 20                                                        ______________________________________                                        Urease Activity                                                                               Absorbance 535 nm                                             Time (min)      CLEC urease                                                                             Soluble urease                                      ______________________________________                                        1     0             0.843     0.901                                             2 1 0.729 0.809                                                               3 3 0.525 0.488                                                               4 5 0.311 0.170                                                               5 10   0.063 0.022                                                            6 15  0.036 0.000                                                           ______________________________________                                    

Enzymatic Activity of Soluble and CLEC Urease

The enzymatic activity of soluble and CLEC urease was assayedspectrophotometrically (Table 20 and FIG. 13) by hydrolysis of thesubstrate urea at room temperature (Table 20 and FIG. 1). Initialsubstrate concentration was 0.39 ug/ml (6.5×10⁻⁸ M) as determined byBradford protein determination. The reaction volume was 5 ml. Ureahydrolysis was quantified calorimetrically using the Sigma diagnosticsblood urea nitrogen kit (procedure No. 535) according to themanufacturer's instructions. Absorbance was fitted to a first order rateequation and kcat/Km was calculated by dividing the fitted value byenzyme concentration as described in Example 2.

                  TABLE 21                                                        ______________________________________                                        Urease pH curve                                                                             % Maximum Activity                                              pH            CLEC urease                                                                             Soluble urease                                        ______________________________________                                        1      5           0         0                                                  2 6 62 51                                                                     3 7 100  100                                                                  4 8 97 91                                                                     5 9 74 69                                                                   ______________________________________                                    

pH Dependence and Stability

The pH optimum and stability of the soluble urease were compared to thatof urease CLECs by cleavage of urea at the indicated pH (Table 21 andFIG. 14). Both CLEC and soluble urease show a similar pH profile, havingoptimum activity at pH 7.

                  TABLE 22                                                        ______________________________________                                        Urease thermal stability                                                                      % Maximum Activity                                            Time (days)     CLEC urease                                                                             Soluble urease                                      ______________________________________                                        1      0.000        100       100.0                                             2 0.042  86.0                                                                 3 0.083 97 71.5                                                               4 0.166  61.5                                                                 5 0.330  52.0                                                                 6 0.416 94 42.0                                                               7 0.666  10.5                                                                 8 1.000 96 6.5                                                                9 2.000 93                                                                    10  3.000 91                                                                  11  4.000 87                                                                ______________________________________                                    

Thermal Stability at 55° C.

The activity of soluble and CLEC urease was measured followingincubation at 55° C. Soluble and CLEC urease were incubated withstirring in 50 mM sodium phosphate buffer pH 7.0 at 55° C. The reactionvolume was one milliliter. Final protein concentration was 10 mg/ml.Aliquots of soluble urease were removed at times 0, 1, 2, 4, 8, 10, 16and 24 hours. Aliquots of CLEC urease were removed at times 0, 2, 10,24, 48, 72 and 96 hours. Enzymatic activity was assayed at roomtemperature as described above (Table 22 and FIG. 15).

                  TABLE 23                                                        ______________________________________                                        Urease protease resistance                                                                    % Maximum Activity                                            Time (days)     CLEC urease                                                                             Soluble urease                                      ______________________________________                                        1      0.000        100       100                                               2 0.014  74                                                                   3 0.028 108 54                                                                4 0.042  29                                                                   5 0.083  13                                                                   6 0.104   3                                                                   7 0.125   0                                                                   8 1.000 106                                                                   9 2.000 107                                                                   10  3.000  94                                                               ______________________________________                                    

Resistance to Exogenous Proteolysis

Assessment of the resistance of the urease CLEC to the action ofprotease was also performed under identical conditions (excepting thesubstitution of 50 mM sodium phosphate pH 7.5 for 50 mM Tris pH 7.5 asthe base buffer) as described for thermolysin (Example 2). Activity ofthe soluble and CLEC enzyme, following incubation with protease, wasassayed as described above (Table 23 and FIG. 16).

                  TABLE 24                                                        ______________________________________                                        Urease stability in organic solvents                                                          % Maximum Activity                                                          CLEC urease                                                                           Soluble urease                                          ______________________________________                                        1       Acetonitrile                                                                              93        40                                                2 Dioxane 95 45                                                               3 Acetone 90 61                                                               4 THF 82 13                                                                 ______________________________________                                    

Stability in Organic Solvents

Urease CLECs and soluble urease were incubated for one hour at 40° C. in50% (v/v) solutions of the indicated organic solvents (as described inExample 2) (Table 24). Following incubation, enzyme activity was assayedby measuring urea hydrolysis as described above.

                  TABLE 25                                                        ______________________________________                                        Urease CLEC catalytic activity in sera                                                          Absorbance 535 nm                                           Time (min)        Buffer   Sera                                               ______________________________________                                        1       0             1.066    1.059                                            2  3 0.769 0.714                                                              3  5 0.672 0.644                                                              4 10 0.537 0.489                                                              5 20 0.206 0.225                                                              6 30 0.146 0.140                                                              7 45 0.030 0.025                                                            ______________________________________                                    

Enzymatic Activity of Urease CLECs in Sera

The catalytic activity of urease CLECs in sera was compared to itsactivity in 50 mM Tris buffer pH 7.0. Urease CLECs were incubated inmouse sera for 16 hours at room temperature. Following incubation, ureawas added to the sera to a concentration of 0.39 ug/ml. The rate of ureahydrolysis in sera was measured with a Sigma diagnostics blood ureanitrogen kit following the manufacturer's instructions. CLEC ureaseactivity in 50 mM sodium phosphate pH 6.0 following 16 hours incubationin phosphate buffer at room temperature was used as a control. CLECurease catalytic activity in sera, a biologically relevant medium, wascomparable to activity in aqueous buffer (Table 25 and FIG. 17).

EXAMPLE 11 Crystallization and Crosslinking of Candida CylindraceaLipase and Assessment of Characteristics of the Resulting Product

Crystallization of Lipase

Lyophilized Candida cylindracea lipase (9.98 mg dry weight) (BoehringerMannheim) was dissolved in 0.5 ml of deionized water. Final proteinconcentration was 6 mg/ml. To induce crystallization, the lipase wasdialysed against a low salt buffer. Five buffer conditions producedcrystals suitable for CLEC formulation: Condition 1, Dialysis against 5mM sodium phosphate buffer pH 6; Condition 2, Dialysis against 5 mMsodium phosphate buffer pH 7; Condition 3, Dialysis against 5 mM sodiumphosphate pH 8; Condition 4, Dialysis against 20 mM Tris HCl pH 6.8;Condition 5, Dialysis against 20 mM Tris HCl pH 6.8 containing 1 mMCaCl₂ and 1 mM MgCl₂. All dialysis conditions produced a showering oflipase crystals; thin square plates 0.1 mm-0.05 mm in size.Crystallization was completed in six hours. Crystals were recovered bycentrifugation as previously described. Lipase crystals were washed tentimes with 20 mM buffer pH 6.8. Following washing, crystals were againrecovered by centrifugation.

Cross-Linking of Candida Cylindracea Lipase Crystals

As disclosed here, lipase crystals were cross-linked in a solution of7.5% glutaraldehyde and 20 mM Tris HCl pH 6.8 for 30 minutes at roomtemperature. Following cross-linking the crystals were washedexhaustively with 20 mM Tris HCl pH 6.8.

Enzymatic Activity of CLEC Lipase

The catalytic activity of the lipase CLECs was assayed qualitatively byhydrolysis of the calorimetric substrate p-nitrophenyl acetate (Fluka)(M. Semeriva et al. Biochem. Biophys. Res. Comm. 58:808-813 (1974)). Thepresence of catalytic activity is indicated by the appearance of ayellow color in the assay solution. Several lipase CLECs were placed ina 100 μl solution of 3.12 mM p-nitrophenyl acetate containing 4%acetonitrile in 80 mM Tris HCl pH 7.5 at room temperature. To monitorspontaneous hydrolysis of the substrate, a negative control containingassay mix and buffer was prepared simultaneously. Soluble lipase wasused as a positive control. Lipase CLECs were found to retainsignificant activity.

EXAMPLE 11 Porcine Pancreatic Lipase Purification

The purification was based in large part on the published procedure ofRoberts et al., Lipids 20:42-45 (1985). The following are thedifferences between the purification procedure used herein and thepublished protocol.

1. The initial extraction buffer, "buffer A" in the publication, doesnot contain sodium azide.

2. The procedure was followed exactly as published up through thebutanol extraction and then the following additional steps were taken.

A. The "creamy interface" resultant from the butanol extraction wasresuspended in 150 ml publication "buffer B".

B. The butanol extraction was repeated as described in the publication.

Two butanol extractions were performed instead of one.

3. The published procedure was followed up through the overnightdialysis and centrifugation. No column chromatography was run.

4. This procedure is run at 4× the published scale.

5. The final product is frozen at -20° C.

Original Crystallization

1. The frozen lipase was thawed and dialyzed vs. 5 mM cacodylate pH 7.0,3.3 mM CaCl₂. Dialysate was centrifuged at 5000×G for 5 minutes.Supernatant was sequentially concentrated and diluted with 5 mMcacodylate pH 7.0 until the following conditions were reached:

Protein concentration=97 mg/ml

CaCl₂ concentration=0.2 mM

Na taurodeoxycholate concentration=0.5 mM

2. Vapor diffusion.

Reservoir: 1.0 ml 10% (v/v) PEG 400 in H₂ O

Drop: 5 ul protein from step #1+5 ul reservoir

A large number of rods were observed overnight under these conditions.

Batch Crystallization

1. Purified lipase was allowed to sit undisturbed in the cold room underthe following conditions:

Protein concentration=46 mg/ml

Buffer=5 mM cacodylate pH 7.0, 0.02 mm CaCl₂, 0.5 mM Nataurodeoxycholate

Lipase sat from Dec. 6, 1991 to Jan 7, 1992, when a large number of rodshaped crystals were observed.

2. These crystals were washed in 20% (v/v) PEG 400 and crushed. Thefollowing seeding was performed:

100 ul purified lipase in 5 mM cacodylate, pH7.0 concentration=77 mg/ml

5 ul PEG 400

5 ul seeds

The lipase rods were observed to "crash out" almost instantly at roomtemperature.

Cross-Linking (Original Conditions)

Wash crystals in 30% PEG 400, 5 mM cacodylate, pH 7.0

Crosslink at room temperature in 3.3% glutaraldehyde for 1.5 hour

Wash in 20 mM Tris pH 8.0.

EXAMPLE 12

We have observed that crystal nucleation and growth will occur on avariety of forms and material surfaces. The following study wasperformed with the protease thermolysis.

Stainless steel forceps, glass beads, string and chromatography matrix,were each immersed in a crystallization solution of thermolysis proteinprepared as described in U.S. patent application Ser. No. 07/720,237 asfiled on Jun. 4, 1991 and allowed to incubate overnight at roomtemperature. Incubation resulted in the attachment of a coating ofthermolysis crystals on all surfaces in contact with the proteinsolution.

The use of attached lyophilized (dried) or unlyophilized, crosslinked oruncrosslinked crystalline material as described in U.S. patentapplication Ser. No. 07/720,237, should significantly benefit the use ofproteins and related compounds in industry and science, by permittingthe coating of a variety of materials with any crystalline material ofinterest. Crystallization occurs at a density approaching theoreticalpacking limits for a given molecule. This phenomena may be of import inany application which requires a high density of protein per unitvolume, such as bioelectronics, materials science and biosensors.Moreover, crystal deposition may be directed and controlled by a numberof means including pretreatment or charging of the crystallizationsurface, limiting the rate and extent of crystallization and definingindividual crystal size. A given crystal or crystalline surface couldalso be used as a site of attachment for a different crystallinematerial. For example, two enzymes, one of which generates a cofactor orsubstrate for a different enzyme, could both be effectivelyco-crystallized in the same functional space. Finally, isolated orclustered crystals or a crystalline layer or layers of the same ordifferent crystalline material could be deposited on, between, or withinvarious surfaces or layers to facilitate application in many fields ofscience and technology.

EXAMPLE 13

We have discovered that crosslinked enzyme crystals (CLECs), asdescribed in U.S. patent application Ser. No. 07/720,237 as filed onJun. 4, 1991 are biocompatible and exhibit enzyme activity in vivo. Thefollowing experiments were performed with thermolysin and asparaginaseCLECs prepared as described in U.S. patent application Ser. No.07/720,237.

Five milligrams of enzymatically active thermolysin CLECs, and fivemilligrams of thermolysin CLECs inactivated by extensive chemicalcrosslinking were injected intraperitonealy into mice. The animalreceiving active CLECs died within two hours. The animal receiving theinactivated CLECs survived several weeks until autopsy, with noobservable effects.

In a preliminary experiment, asparaginase CLECs in doses of 1 to 200 IUwere injected intraperitonealy into control and lymphoma induced mice.There was no difference in survival time between asparaginase treatedand control (untreated) lymphoma induced mice. Asparaginase CLECs didnot demonstrate any toxic effects.

There is significant potential to employ enzymes as therapeutics: forexample, to remove toxins, drugs and metabolites in the treatment ofseveral conditions, including acute lymphoid leukemias and inborn errorsof metabolism. Immunogenicity and short half-life currently limit theuse of proteins, peptides and related molecules as therapeutics.Crystalline uncrosslinked or crosslinked, proteins and peptides resistinactivation by proteolysis and acids, and are likely to benonimmunogenic. CLECs also demonstrate chromatographic properties makingthem suitable for use in extracorporeal devices for treatment ofdiseases such as chronic renal failure. In addition, CLECs areinsoluble, making the use of entrapment and implantation of the CLECsattractive. The size of the crystal can also be tightly controlled,making oral and injectable therapeutic delivery routes feasible.

EXAMPLE 14

We have discovered that crosslinked enzyme crystals (CLECs) as describedin U.S. patent application Ser. No. 07/720,237 as filed on Jun. 4, 1991demonstrate chromatographic properties. The following procedure wasperformed with CLECs of the protease thermolysin:

A 15 microliter volume of thermolysin CLECs prepared as described inU.S. patent application Ser. No. 07/720,237 was placed in a small columnforming a bed volume of 2 mm×200 mm. One hundred milliliters of waterwere passed through the column at a flow rate of 25 ml per hour. Proteinassay of the flow through indicated that no protein was present. Twentymilliliters of a thermolysin spectrophotometric substrate as describedin U.S. patent application Ser. No. 07/720,237 was then passed throughthe column at a flow rate of 25 ml per hour. Spectrophotometric analysisof the flow through indicated that complete hydrolysis of the substratehad occurred. Further, the CLEC enzyme column bed showed no change inflow rate following a through put of greater than 700 bed volumes.

This is a novel observation to the best of our knowledge, and shouldsignificantly benefit the use of enzymes and proteins in industry andscience by, among other things facilitating the use of CLEC proteins inapplications where chromatographic properties are advantageous.

EXAMPLE 15

We have observed that crystal growth and hence size can be controlled bya number of different methods. We have demonstrated this on both themilligram and tens of gram scale. The following protocol was performedwith the protease thermolysin.

Purified thermolysin crystals, 90% thermolysin enzyme protein by weight,were solubilized in a crystallization solution of 30% DMSO and 0.25 Mcalcium acetate pH 6.5. Thermolysin crystallization was induced byreducing the DMSO concentration of the crystallization solution asdescribed in U.S. patent application Ser. No. 07/720,237 as filed onJun. 4, 1991. Crystal nucleation, growth and size were reproduciblycontrolled through manipulation of the crystallization solution proteinconcentration and magnitude of DMSO dilution. Two micron crystals wereprepared by the dilution of a 100 mg/ml thermolysin solution with 5volumes of calcium buffer where as 100 micron crystals were obtained bydilution of a 50 mg/ml solution with 3 volumes of buffer. Agitation,temperature, the addition of seed crystals, and combinations of theseand other crystallization parameters may be used to reproducibly controlcrystal size.

These observations should significantly benefit the use of enzymes,proteins, and peptides in science industry and medicine. For examplecrosslinked enzyme crystals (CLECs), as described in U.S. patentapplication Ser. No. 07/720,237, could be designed for specificindustrial applications to optimize catalytic efficiency and ease ofrecovery. Protein crystal applications in the fields of biosensors,bioelectronics and materials science may also derive benefit fromcrystals of defined dimensions. Therapeutic applications may alsorequire a specific crystal size, for example crystals of less than tenmicrons may permit injection and oral delivery of crosslinked oruncrosslinked proteins, peptides and related molecules.

EXAMPLE 16

We have observed that protein crystals are stable and catalyticallyactive for extended periods of time in buffered organic solvents, andaqueous solutions having sufficient osmolarity and buffering capacity tostabilize the crystalline structure. Further, altering the environmentof the stabilized crystal by, for example, pH change, temperature changeor osmotic pressure change, will induce the solubilization of thecrystal to active soluble protein molecules. The following experimentswere performed with the protease subtilisin carlsberg and Candidacylindreacea lipase.

A preparation of crystalline subtilisin carlsberg remained catalyticallyactive and in crystal form at room temperature in a nonsterile aqueoussolution of 17% sodium sulfate at pH 5.9 for 30 days. The crystals werethen solubilized to active monomers by shifting the osmolarity of thecrystal environment from 17% sodium sulfate <0.5% sodium sulfate pH 5.9or to 10 mM sodium phosphate pH 5.9. Alternatively the crystals weresolubilized by maintaining the osmotic environment and shifting the pHfrom acidic (pH 5.9) to basic (9.0) conditions. The rate ofsolubilization could be controlled by varying protein concentrationand/or the magnitude of change in one or more parameters of the crystalenvironment. Spectrophotometric activity assay of the solubilizedcrystals showed that the formerly crystalline enzyme had activitycomparable to that observed for a fresh preparation of soluble enzyme.

Candida cylindracea lipase crystals were used to catalyze theesterification of an alcohol in a buffered organic solvent. The enzymecrystals maintained their structural integrity during the 72 hourreaction. Approximately 10,000 units of candida lipase crystals weredried with phosphate buffered ethano pH 7.0. The lipase crystals werethen added to a 2 ml reaction containing 130 mM dl menthol and 100 mM5-phenylvaleric acid in phosphate buffer saturated isooctane pH 7.0. Thereaction was incubated at 30° C. with shaking at 150 rpm for 72 hours.Thin layer chromatography of the reaction mix demonstrated the presenceof the esterification product, menthol phenyl valerate.

The application of these observations should greatly benefit the use ofenzymes in industry, science and medicine, by permitting thestabilization of enzymes, proteins, peptides and related molecules, incrystal form, followed by the controlled dissociation of the crystallattice releasing the active free molecule.

EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain, usingno more than routine experimentation, many equivalents to the specificmaterials and components described herein. Such equivalents are intendedto be encompassed in the scope of the following claims.

We claim:
 1. A biosensor for detecting an analyte of interest in afluid, comprising:(a) a protein crystal crosslinked with amultifunctional crosslinking agent, said crosslinked protein crystalhaving resistance to exogenous proteolysis, such that said crosslinkedprotein crystal retains at least 91% of its stability, as measured interms of degradation, after incubation for three hours in the presenceof a concentration of Pronase™ that causes the soluble uncrosslinkedform of the protein that is crystallized to form said protein crystalthat is crosslinked to lose at least 94% of its stability, as measuredin terms of degradation, under the same conditions, wherein said proteinhas the activity of acting on the analyte of interest or on a reactantin a reaction in which the analyte of interest participates; (b) aretaining means for said crosslinked protein crystal, said retainingmeans consisting of a material which allows contact between saidcrosslinked protein crystal and a fluid, said fluid containing either(1) the analyte upon which said protein acts or (2) a reactant in areaction in which the analyte participates; and (c) a signal transducerwhich produces a signal in the presence of the analyte.
 2. A biosensorfor detecting an analyte of interest in a fluid, comprising:(a) anenzyme crystal crosslinked with a multifunctional crosslinking agent,said crosslinked enzyme crystal having resistance to exogenousproteolysis, such that said crosslinked enzyme crystal retains at least91% of its initial activity after incubation for three hours in thepresence of a concentration of Pronase™ that causes the solubleuncrosslinked form of the enzyme that is crystallized to form saidenzyme crystal that is crosslinked to lose at least 94% of its initialactivity under the same conditions, wherein said enzyme has the activityof acting on the analyte of interest or on a reactant in a reaction inwhich the analyte of interest participates; (b) a retaining means forsaid crosslinked enzyme crystal, said retaining means consisting of amaterial which allows contact between said crosslinked enzyme crystaland a fluid, said fluid containing either (1) the analyte upon whichsaid enzyme acts or (2) a reactant in a reaction in which the analyteparticipates; and (c) a signal transducer which produces a signal in thepresence of the analyte.
 3. The biosensor according to claim 1 or 2,wherein said crystal is a microcrystal.
 4. The biosensor according toclaim 3, wherein said microcrystal has a cross-section of 10⁻¹ mm orless.
 5. The biosensor according to claim 2, wherein said enzyme is ahydrolase.
 6. The biosensor according to claim 5, wherein said hydrolaseis selected from the group consisting of thermolysin, elastase,asparaginase, lysozyme and urease.
 7. The biosensor according to claim5, wherein said hydrolase is an esterase.
 8. The biosensor according toclaim 5, wherein said hydrolase is a lipase.
 9. The biosensor accordingto claim 1 or 2, wherein said biosensor further comprises means fordetecting the activity of said protein or enzyme on said analyte orreactant.
 10. The biosensor according to claim 9, wherein said means fordetecting the activity of said protein or enzyme on said analyte orreactant is selected from the group consisting of pH electrodes, lightsensing devices, heat sensing devices and means for detecting electricalcharge.
 11. The biosensor according to claim 1 or 2, wherein saidanalyte is selected from the group consisting of glucose, creatinine,urea, lactate, glucose-6-phosphate, sucrose, ATP, ethanol, acetic acid,formic acid, cholesterol, uric acid, methotrexate, carbon dioxide, aminoacids, phosphates, penicillin, nitrates, nitrites, sulphates andsuccinate.
 12. An extracorporeal device for use in altering a componentof a fluid, comprising:(a) a protein crystal crosslinked with amultifunctional crosslinking agent, said crosslinked protein crystalhaving resistance to exogenous proteolysis, such that said crosslinkedprotein crystal retains at least 91% of its stability, as measured interms of degradation, after incubation for three hours in the presenceof a concentration of Pronase™ that causes the soluble uncrosslinkedform of the protein that is crystallized to form said protein crystalthat is crosslinked to lose at least 94% of its stability, as measuredin terms of degradation, under the same conditions, wherein said proteinhas the activity of acting on the component or on a reactant in areaction in which the component participates; and (b) a retaining meansfor said crosslinked protein crystal, said retaining means consisting ofa material which allows contact between said crosslinked protein crystaland a fluid, said fluid containing either (1) the component upon whichsaid protein acts or (2) a reactant in a reaction in which the componentparticipates.
 13. An extracorporeal device for use in altering acomponent of a fluid, comprising:(a) an enzyme crystal crosslinked witha multifunctional crosslinking agent, said crosslinked enzyme crystalhaving resistance to exogenous proteolysis, such that said crosslinkedenzyme crystal retains at least 91% of its initial activity afterincubation for three hours in the presence of a concentration ofPronase™ that causes the soluble uncrosslinked form of the enzyme thatis crystallized to form said enzyme crystal that is crosslinked to loseat least 94% of its initial activity under the same conditions, whereinsaid enzyme has the activity of acting on the component or on a reactantin a reaction in which the component participates; and (b) a retainingmeans for said crosslinked enzyme crystal, said retaining meansconsisting of a material which allows contact between said crosslinkedenzyme crystal and a fluid, said fluid containing either (1) thecomponent upon which said enzyme acts or (2) a reactant in a reaction inwhich the component participates.
 14. The extracorporeal deviceaccording to claim 12 or 13, wherein said crystal is a microcrystal. 15.The extracorporeal device according to claim 14, wherein saidmicrocrystal has a cross-section of 10⁻¹ mm or less.
 16. Theextracorporeal device according to claim 13, wherein said enzyme is ahydrolase.
 17. The extracorporeal device according to claim 16, whereinsaid hydrolase is selected from the group consisting of thermolysin,elastase, asparaginase, lysozyme and urease.
 18. The extracorporealdevice according to claim 16, wherein said hydrolase is an esterase. 19.The extracorporeal device according to claim 16, wherein said hydrolaseis a lipase.
 20. The extracorporeal device according to claim 12 or 13,wherein said device further comprises a deheparinization device locatedat an effluent of the extracorporeal device.
 21. The extracorporealdevice according to claim 20, wherein said deheparinization device isselected from the group consisting of continuous arteriovenoushemofiltration devices and extracorporeal membrane oxygenators.
 22. Theextracorporeal device according to claim 12 or 13, wherein saidcomponent is selected from the group consisting of heparin, bilirubin,methotrexate, amino acids, urea and ammonia.
 23. The extracorporealdevice according to claim 12 or 13, wherein said retaining means is aporous material on which said crosslinked protein or enzyme crystal isretained or a tube in which said crosslinked protein or enzyme crystalis present.
 24. A method for detecting the presence of a substance in asample comprising the steps of:(a) contacting the sample with a proteinwhich is a protein crystal crosslinked with a multifunctionalcrosslinking agent, said crosslinked protein crystal having resistanceto exogenous proteolysis, such that said crosslinked protein crystalretains at least 91% of its stability, as measured in terms ofdegradation, after incubation for three hours in the presence of aconcentration of Pronase™ that causes the soluble uncrosslinked form ofthe protein that is crystallized to form said protein crystal that iscrosslinked to lose at least 94% of its stability, as measured in termsof degradation, under the same conditions, wherein said protein has theactivity of acting on the substance or on a reactant in a reaction inwhich the substance participates to cause a detectable change, underconditions and for a time sufficient to permit said activity; and (b)using a detection means to detect the detectable change.
 25. A methodfor detecting the presence of a substance in a sample comprising thesteps of:(a) contacting the sample with an enzyme which is an enzymecrystal crosslinked with a multifunctional crosslinking agent, saidcrosslinked enzyme crystal having resistance to exogenous proteolysis,such that said crosslinked enzyme crystal retains at least 91%, of itsinitial activity after incubation for three hours in the presence of aconcentration of Pronase™ that causes the soluble uncrosslinked form ofthe enzyme that is crystallized to form said enzyme crystal that iscrosslinked to lose at least 94% of its initial activity under the sameconditions, wherein said enzyme has the activity of acting on thesubstance or on a reactant in a reaction in which the substanceparticipates to cause a detectable change, under conditions and for atime sufficient to permit said activity; and (b) using a detection meansto detect the detectable change.
 26. The method according to claim 24 or25, wherein said crystal is a microcrystal.
 27. The method according toclaim 26, wherein said microcrystal has a cross-section of 10⁻¹ mm orless.
 28. The method according to claim 25, wherein said enzyme is ahydrolase.
 29. The method according to claim 28, wherein said hydrolaseis selected from the group consisting of thermolysin, elastase,asparaginase, lysozyme and urease.
 30. The method according to claim 28,wherein said hydrolase is an esterase.
 31. The method according to claim28, wherein said hydrolase is a lipase.
 32. The method according toclaim 24 or 25, wherein the detectable change is selected from the groupconsisting of change in pH, production of light, production of heat andchange in electrical potential.